Part I
Foundations of Biochemistry
Facing page: Supernova SN 1987a (the bright "star" at the lower right) resulted from the explosion of a blue supergiant star in the Large Magellanic Cloud, a galaxy near the Milky Way. Energy released by nuclear explosions in such supernovae brought about the fusion of simple atomic nuclei, forming the more complex elements of which the earth, its atmosphere, and all living things are composed.

Fifteen to twenty billion years ago the universe arose with a cataclysmic explosion that hurled hot, energy-rich subatomic particles into all space. Within seconds, the simplest elements (hydrogen and helium) were formed. As the universe expanded and cooled, galaxies condensed under the influence of gravity. Within these galaxies, enormous stars formed and later exploded as supernovae, releasing the energy needed to fuse simpler atomic nuclei into the more complex elements. Thus were produced, over billions of years, the chemical elements found on earth today. Biochemistry asks how the thousands of different biomolecules formed from these elements interact with each other to confer the remarkable properties of living organisms.

In Part I we will summarize the biological and chemical background to biochemistry. Living organisms operate within the same physical laws that apply to all natural processes, and we begin by discussing those laws and several axioms that flow from them (Chapter 1). These axioms make up the molecular logic of life. They define the means by which cells transform energy to accomplish work, catalyze the chemical transformations that typify them, assemble molecules of great complexity from simpler subunits, form supramolecular complexes that are the machinery of life, and store and pass on the instructions for the assembly of all future generations of organisms from simple, nonliving precursors.

Cells, the units of all living organisms, share certain features; but the cells of different organisms, and the various cell types within a single organism, are remarkably diverse in structure and function. Chapter 2 is a brief description of the common features and the diverse specializations of cells, and of the evolutionary processes that lead to such diversity.

Nearly all of the organic compounds from which living organisms are constructed are products of biological activity. These biomolecules were selected during the course of biological evolution for their fitness in performing specific biochemical and cellular functions. The biomolecules can be characterized and understood in the same terms that apply to the molecules of inanimate matter: the types of bonds between atoms, the factors that contribute to bond formation and bond strength, the three-dimensional structure of molecules, and chemical reactivities. Three-dimensional structure is especially important in biochemistry; the specificity of biological interactions, such as those between enzyme and substrate, antibody and antigen, hormone and receptor, is achieved by close steric complementarity between molecules. Prominent among the forces that stabilize three-dimensional

structure are noncovalent interactions, individually weak but with significant cumulative effects on the structure of biological macromolecules. Chapter 3 provides the chemical basis for later discussions of the structure, catalysis, and metabolic interconversions of individual classes of biomolecules.

Water is the medium in which the first cells arose, and the solvent in which most biochemical transformations occur. The properties of water have shaped the course of evolution and exert a decisive influence on the structure of biomolecules in aqueous solution. Many of the weak interactions within and between biomolecules are strongly affected by the solvent properties of water. Even water-insoluble components of cells, such as membrane lipids, interact with each other in ways dictated by the polar properties of water. In Chapter 4 we consider the properties of water, the weak noncovalent interactions that occur in aqueous solutions of biomolecules, and the ionization of water and of solutes in aqueous solution.

These initial chapters are intended to provide a chemical backdrop for the later discussions of biochemical structures and reactions, so that whatever your background in chemistry or biology, you can immediately begin to follow, and to enjoy, the action.
 Figure 1–1 Some characteristics of living matter.
(a) Microscopic complexity and organization are apparent in this thin section of vertebrate muscle tissue, viewed with the electron microscope. (b) The lion uses organic compounds obtained by eating other animals to fuel intense bursts of muscular activity. The zebra derives energy from compounds in the plants it consumes; the plants derive their energy from sunlight. (c) Biological reproduction occurs with near-perfect fidelity.
 Erwin Schrödinger
 Figure 1–2 Diverse living organisms share common chemical features. The eagle, the oak tree, the soil bacterium, and the human share the same basic structural units (cells), the same kinds of macromolecules (DNA, RNA, proteins) made up of the same kinds of monomeric subunits (nucleotides, amino acids), the same pathways for synthesis of cellular components, and the same genetic code and evolutionary ancestors.
 Figure 1–3 Monomeric subunits in linear sequences can spell infinitely complex messages. The number of different sequences possible (S) depends on the number of different kinds of subunits (N) and the length of the linear sequence (L): S = NL. For polymers the size of proteins (L ≈ 1,000), S is very large, and for nucleic acids, for which L may be many millions, S is astronomical.
Chapter 1
The Molecular Logic of Life
The Chemical Unity of Diverse Living Organisms

Living organisms are composed of lifeless molecules. When these molecules are isolated and examined individually, they conform to all the physical and chemical laws that describe the behavior of inanimate matter. Yet living organisms possess extraordinary attributes not shown by any random collection of molecules. In this chapter, we first consider the properties of living organisms that distinguish them from other collections of matter. After arriving at a broad definition of life, we can describe a set of principles that characterize all living organisms. These principles underlie the organization of organisms and the cells that make them up, and they provide the framework for this book. They will help you to keep the larger picture in mind while exploring the illustrative examples presented in the text.

Living Matter Has Several Characteristics

What distinguishes all living organisms from all inanimate objects? First, they are structurally complicated and highly organized. They possess intricate internal structures (Fig. 1–1a) and contain many kinds of complex molecules. By contrast, the inanimate matter in our environment – clay, sand, rocks, seawater – usually consists of mixtures of relatively simple chemical compounds.

Second, living organisms extract, transform, and use energy from their environment (Fig. 1–1b), usually in the form of either chemical nutrients or the radiant energy of sunlight. This energy enables living organisms to build and maintain their own intricate structures and to do mechanical, chemical, osmotic, and other types of work. By contrast, inanimate matter does not use energy in a systematic way to maintain structure or to do work. Inanimate matter tends to decay toward a more disordered state, to come to equilibrium with its surroundings.

The third and most characteristic attribute of living organisms is the capacity for precise self-replication and self-assembly (Fig. 1–1c), a property that can be regarded as the quintessence of the living state. A single bacterial cell placed in a sterile nutrient medium can give rise to a billion identical "daughter" cells in 24 hours. Each of the cells contains thousands of different molecules, some extremely complex; yet each bacterium is a faithful copy of the original, constructed entirely from information contained within the genetic material of the original cell. By contrast, mixtures of inanimate matter show no capacity to grow and reproduce in forms identical in mass, shape, and internal structure, generation after generation.

The ability to self-replicate has no true analog in the nonliving world, but there is an instructive analogy in the growth of crystals in saturated solutions. Crystallization produces more material identical in lattice structure with the original "seed" crystal. Crystals are much less complex than the simplest living organisms, and their structure is static, not dynamic as are living cells. Nonetheless, the ability of crystals to "reproduce" themselves led the physicist Erwin Schrödinger to propose in his famous essay "What Is Life?" that the genetic material of cells must have some of the properties of a crystal. Schrödinger’s 1944 notion (years before the modern understanding of gene structure was achieved) describes rather accurately some of the properties of deoxyribonucleic acid, the material of genes.

Each component of a living organism has a specific function. This is true not only of macroscopic structures such as leaves and stems or hearts and lungs, but also of microscopic intracellular structures such as the nucleus or chloroplast. Even individual chemical compounds in cells have specific functions. The interplay among the chemical components of a living organism is dynamic; changes in one component cause coordinating or compensating changes in another, with the result that the whole ensemble displays a character beyond that of the individual constituents. The collection of molecules carries out a program, the end result of which is the reproduction of the program and the self-perpetuation of that collection of molecules.

Biochemistry Seeks to Explain Life in Chemical Terms

The molecules of which living organisms are composed conform to all the familiar laws of chemistry, but they also interact with each other in accordance with another set of principles, which we shall refer to collectively as the molecular logic of life. These principles do not involve new or as yet undiscovered physical laws or forces. Instead, they are a set of relationships characterizing the nature, function, and interactions of biomolecules.

If living organisms are composed of molecules that are intrinsically inanimate, how do these molecules confer the remarkable combination of characteristics we call life? How is it that a living organism appears to be more than the sum of its inanimate parts? Philosophers once answered that living organisms are endowed with a mysterious and divine life force, but this doctrine (vitalism) has been firmly rejected by modern science. The basic goal of the science of biochemistry is to determine how the collections of inanimate molecules that constitute living organisms interact with each other to maintain and perpetuate life. Although biochemistry yields important insights and practical applications in medicine, agriculture, nutrition, and industry, it is ultimately concerned with the wonder of life itself.

Chemical Unity Underlies Biological Diversity

A massive oak tree, an eagle that soars above it, and a soil bacterium that grows among its roots appear superficially to have very little in common. However, a hundred years of biochemical research has revealed that living organisms are remarkably alike at the microscopic and chemical levels (Fig. 1–2). Biochemistry seeks to describe in molecular terms those structures, mechanisms, and chemical processes shared by all organisms and to discover the organizing principles that underlie life in all of its diverse forms.

Although there is a fundamental unity to life, it is important to recognize at the outset that very few generalizations about living organisms are absolutely correct for every organism under every condition. The range of habitats in which organisms live, from hot springs to Arctic tundra, from animal intestines to college dormitories, is matched by a correspondingly wide range of specific biochemical adaptations. These adaptations are integrated within the fundamental chemical framework shared by all organisms. Although generalizations are not perfect, they remain useful. In fact, exceptions often illuminate scientific generalizations.

All Macromolecules Are Constructed from a Few Simple Compounds

Most of the molecular constituents of living systems are composed of carbon atoms covalently joined with other carbon atoms and with hydrogen, oxygen, or nitrogen. The special bonding properties of carbon permit the formation of a great variety of molecules. Organic compounds of molecular weight (Mr) less than about 500, such as amino acids, nucleotides, and monosaccharides, serve as monomeric subunits of proteins, nucleic acids, and polysaccharides, respectively. A single protein molecule may have 1,000 or more amino acids, and deoxyribonucleic acid has millions of nucleotides.

Each cell of the bacterium Escherichia coli (E. coli) contains more than 6,000 different kinds of organic compounds, including about 3,000 different proteins and a similar number of different nucleic acid molecules. In humans there may be tens of thousands of different kinds of proteins, as well as many types of polysaccharides (chains of simple sugars), a variety of lipids, and many other compounds of lower molecular weight.

To purify and to characterize thoroughly all of these molecules would be an insuperable task were it not for the fact that each class of macromolecules (proteins, nucleic acids, polysaccharides) is composed of a small, common set of monomeric subunits. These monomeric subunits can be covalently linked in a virtually limitless variety of sequences (Fig. 1–3), just as the 26 letters of the English alphabet can be arranged into a limitless number of words, sentences, or books.

Deoxyribonucleic acids (DNA) are constructed from only four different kinds of simple monomeric subunits, the deoxyribonucleotides, and ribonucleic acids (RNA) are composed of just four types of ribonucleotides. Proteins are composed of 20 different kinds of amino acids. The eight kinds of nucleotides from which all nucleic acids are built and the 20 different kinds of amino acids from which all proteins are built are identical in all living organisms.

Most of the monomeric subunits from which all macromolecules are constructed serve more than one function in living cells. The nucleotides serve not only as subunits of nucleic acids, but also as energy-carrying molecules. The amino acids are subunits of protein molecules, and also precursors of hormones, neurotransmitters, pigments, and many other kinds of biomolecules.


From these considerations we can now set out some of the principles in the molecular logic of life:

All living organisms have the same kinds of monomeric subunits.

There are underlying patterns in the structure of biological macromolecules.

The identity of each organism is preserved by its possession of distinctive sets of nucleic acids and of proteins.
 Figure 1–4 Living organisms are not at equilibrium with their surroundings. Death and decay restore the equilibrium. During growth, energy from food is used to build complex molecules and to concentrate ions from the surroundings. When the organism dies, it loses its ability to derive energy from food. Without energy, the dead body cannot maintain concentration gradients; ions leak out. Inexorably, macromolecular components decay to simpler compounds. These simple compounds serve as nutritional sources for phytoplankton, which are then eaten by larger organisms. (By convention, square brackets denote concentration – in this case, of ionic species.)
 Figure 1–5 A dynamic steady state results when the rate of appearance of a cellular component is exactly matched by the rate of its disappearance. In (a), a protein (hemoglobin) is synthesized, then degraded. In (b), glucose derived from food (or from carbohydrate stores) enters the bloodstream in some tissues (intestine, liver), then leaves the blood to be consumed by metabolic processes in other tissues (heart, brain, skeletal muscle). In this scheme, r1, r2, etc., represent the rates of the various processes. The dynamic steady-state concentrations of hemoglobin and glucose are maintained by complex mechanisms regulating the relative rates of the processes shown here.
 Figure 1–6 (Top) The downward motion of an object releases potential energy that can do work. The potential energy made available by spontaneous downward motion (an exergonic process, represented by the pink box) can be coupled to the upward movement of another object (an endergonic process, represented by the blue box). (Bottom) A spontaneous (exergonic) chemical reaction (B→C) releases free energy, which can pull or drive an endergonic reaction (A→B) when the two reactions share a common intermediate, B. The exergonic reaction B→C has a large, negative free-energy change (ΔGB→C), and the endergonic reaction A→B has a smaller, positive free-energy change (ΔGA→B). The free-energy change for the overall reaction A→C is the arithmetic sum of these two values (ΔGA→C). Because the value of ΔGA→C is negative, the overall reaction is exergonic and proceeds spontaneously.
Energy Production and Consumption in Metabolism

Energy is a central theme in biochemistry: cells and organisms depend upon a constant supply of energy to oppose the inexorable tendency in nature for decay to the lowest energy state. The synthetic reactions that occur within cells, like the synthetic processes in any factory, require the input of energy. Energy is consumed in the motion of a bacterium or an Olympic sprinter, in the flashing of a firefly or the electrical discharge of an eel. The storage and expression of information cost energy, without which structures rich in information inevitably become disordered and meaningless. Cells have evolved highly efficient mechanisms for capturing the energy of sunlight, or extracting the energy of oxidizable fuels, and coupling the energy thus obtained to the many energy-consuming processes they carry out.

Organisms Are Never at Equilibrium with Their Surroundings

In the course of biological evolution, one of the first developments must have been an oily membrane that enclosed the water-soluble molecules of the primitive cell, segregating them and allowing them to accumulate to relatively high concentrations. The molecules and ions contained within a living organism differ in kind and in concentration from those in the organism’s surroundings. The cells of a freshwater fish contain certain inorganic ions at concentrations far different from those in the surrounding water (Fig. 1–4). Proteins, nucleic acids, sugars, and fats are present in the fish but essentially absent from the surrounding water, which instead contains carbon, hydrogen, and oxygen atoms only in simpler molecules such as carbon dioxide and water. When the fish dies, its contents eventually come to equilibrium with those of its surroundings.


Molecular Composition Reflects a Dynamic Steady State

Although the chemical composition of an organism may be almost constant through time, the population of molecules within a cell or organism is far from static. Molecules are synthesized and then broken down by continuous chemical reactions, involving a constant flux of mass and energy through the system. The hemoglobin molecules carrying oxygen from your lungs to your brain at this moment were synthesized within the past month; by next month they will have been degraded and replaced with new molecules. The glucose you ingested with your most recent meal is now circulating in your bloodstream; before the day is over these particular glucose molecules will have been converted into something else, such as carbon dioxide or fat, and will have been replaced with a fresh supply of glucose. The amount of hemoglobin and glucose in the blood remains nearly constant because the rate of synthesis or intake of each just balances the rate of its breakdown, consumption, or conversion into some other product (Fig. 1–5). The constancy of concentration does not, therefore, reflect chemical inertness of the components, but is rather the result of a dynamic steady state.

Organisms Exchange Energy and Matter with Their Surroundings

Living cells and organisms must perform work to stay alive and to reproduce themselves. The continual synthesis of cellular components requires chemical work; the accumulation and retention of salts and various organic compounds against a concentration gradient involves osmotic work; and the contraction of a muscle or the motion of a bacterial flagellum represents mechanical work. Biochemistry examines the processes by which energy is extracted, channeled, and consumed, so it is essential to develop an understanding of the fundamental principles of bioenergetics.

Consider the simple mechanical example shown in Figure 1–6. An object at the top of an inclined plane has a certain amount of potential energy as a result of its elevation. It tends spontaneously to slide down the plane, losing its potential energy of position as it approaches the ground. When an appropriate string-and-pulley device is attached to the object, the spontaneous downward motion can accomplish a certain

amount of work, an amount never greater than the change in potential energy of position. The amount of energy actually available to do work (called the free energy) will always be somewhat less than the total change in energy, because some energy is dissipated as the heat of friction. The greater the elevation of the object relative to its final position, the greater the change in energy as it slides downward, and the greater the amount of work that can be accomplished.

In the chemical analog of this mechanical example (Fig. 1–6, bottom), a reactant, B, is converted into a product, C. The compounds B and C each contain a certain amount of potential energy, related to the kind and number of bonds in each type of molecule. This energy is analogous to the potential energy in an elevated object. Some of the energy is available to do work when B is converted into C by a chemical reaction that involves no change in temperature or pressure. This portion of the energy, the free energy, is designated G (for J. Willard Gibbs, who developed much of the theory of chemical energetics), and the change in free energy during the conversion of B to C is ΔG.

We can define a system as all of the reactants and products, the solvent, and the immediate atmosphere – in short, everything within a defined region of space. The system and its surroundings together constitute the universe. If the system exchanges neither matter nor energy with its surroundings, it is said to be closed. The magnitude of the free-energy change for a process proceeding toward equilibrium depends upon how far from equilibrium the system was in its initial state. In the mechanical example, no spontaneous sliding will occur once the object has reached the ground; the object is then at equilibrium with its surroundings, and the free-energy change for sliding along the horizontal surface is zero.

In chemical reactions in closed systems, the process also proceeds spontaneously until equilibrium is reached. The free-energy changeG) for a chemical reaction is a quantitative expression of how far the system is from chemical equilibrium. Reactions that proceed with the release of free energy are exergonic, and because the products of such reactions have less free energy than the reactants, ΔG is negative. Chemical reactions in which the products have more free energy than the reactants are endergonic, and for these reactions ΔG is positive. When all of the chemical species in the system are at equilibrium, the free-energy change for the reaction is zero, and no further net conversion of reactants into products will occur without the input of energy or matter from outside the system.

As in the mechanical example, some of the energy released in a spontaneous process can accomplish work – chemical work in this case. In living systems, as in mechanical processes, part of the total energy change in the chemical reaction is unavailable to accomplish work. Some is dissipated as heat, and some is lost as entropy, a measure of energy due to randomness, which we will define more rigorously later.

How is free energy from a chemical reaction channeled into energy-requiring processes in living organisms? In the mechanical example in Figure 1–6, it is clear that if one sliding object is coupled to another object on another inclined plane, the energy released by the spontaneous downward sliding of one may be harnessed to produce upward motion of the other, a motion that cannot occur spontaneously. This is a direct analogy to a biochemical process in which the energy released in an exergonic chemical reaction can be used to drive another reaction that is endergonic and would not proceed spontaneously. The reactions

in this system are coupled because the product of one (compound B) is a reactant in the other. This coupling of an exergonic reaction with an endergonic one is absolutely central to the free-energy exchanges that occur in all living systems. In biological energy coupling, the simultaneous occurrence of two reactions is not enough. The two reactions must be coupled in the sense of Figure 1–6 (bottom); the two reactions share an intermediate, B.

A living organism is an open system; it exchanges both matter and energy with its surroundings. Living organisms use either of two strategies to derive free energy from their surroundings: (1) they take up chemical components from the environment (fuels), extract free energy by means of exergonic reactions involving these fuels, and couple these reactions to endergonic reactions; or (2) they use energy absorbed from sunlight to bring about exergonic photochemical reactions, to which they couple endergonic reactions.

Living organisms create and maintain their complex, orderly structures at the expense of free energy from their environment.

Exergonic chemical or photochemical reactions are coupled to endergonic processes through shared chemical intermediates, channeling the free energy to do work.
 Figure 1–7 During metabolic transductions, entropy increases as the potential energy of complex nutrient molecules decreases. Living organisms (a) extract energy from their environment, (b) convert some of it into useful forms of energy to produce work, and (c) return some energy to the environment as heat, together with end-product molecules that are less well organized than the starting fuel, increasing the entropy of the universe.
 Figure 1–8 Sunlight is the ultimate source of all biological energy. Thermonuclear reactions in the sun produce energy that is transmitted to the earth as light and converted into chemical energy by plants and certain microorganisms.
 Figure 1–9 The energetic course of a chemical reaction. A high activation barrier, representing the transition state, must be overcome in the conversion of reactants (A) into products (B), even though the products are more stable than the reactants – as indicated by a large, negative free-energy change (ΔG). The energy required to overcome the activation barrier is the activation energy (ΔG). Enzymes catalyze reactions by lowering the activation barrier. They bind the transition-state intermediates tightly, and the binding energy of this interaction efiectively reduces the activation energy from ΔGuncat to ΔGcat. (Note that the activation energy is unrelated to the free-energy change of the reaction, ΔG.)
 Figure 1–10 An enzyme increases the rate of a specific chemical reaction. In the presence of an enzyme specific for the conversion of reactant A into product B, the rate of the reaction may increase a millionfold or more over that of the uncatalyzed reaction. The enzyme is not consumed in the process; one enzyme molecule can act repeatedly to convert many molecules of A to B.
 Figure 1–11 An example of a typical synthetic (anabolic) pathway. In the bacterium E. coli, threonine is converted to isoleucine in five steps, each catalyzed by a separate enzyme. (Only the main reactants and products are shown here.) Threonine, in turn, was synthesized from a simpler precursor. Both threonine and isoleucine are precursors of much larger and more complex molecules: the proteins. (The letters A to F correspond to those in Fig. 1–14.)
Cells and Organisms Interconvert Different Forms of Energy

The first law of thermodynamics, developed from physics and chemistry but fully valid for biological systems as well, describes the energy conservation principle:

In any physical or chemical change, the total amount of energy in the universe remains constant, although the form of the energy may change.

Not until the nineteenth century did physicists discover that energy can be transduced (converted from one form to another), yet living cells have been using that principle for eons. Cells are consummate transducers of energy, capable of interconverting chemical, electromagnetic, mechanical, and osmotic energy with great efficiency (Fig. 1–7). Biological energy transducers differ from many familiar machines that depend on temperature or pressure differences. The steam engine, for example, converts the chemical energy of fuel into heat, raising the temperature of water to its boiling point to produce steam pressure that drives a mechanical device. The internal combustion engine, similarly, depends upon changes in temperature and pressure. By contrast, all parts of a living organism must operate at about the same temperature and pressure, and heat flow is therefore not a useful source of energy. Cells are isothermal, or constant-temperature, systems.

Living cells are chemical engines that function at constant temperature.
The Flow of Electrons Provides Energy for Organisms

Virtually all of the energy transductions in cells can be traced to a flow of electrons from one molecule to another, in the oxidation of fuel or in the trapping of light energy during photosynthesis. This electron flow is "downhill," from higher to lower electrochemical potential; as such, it is formally analogous to the flow of electrons in an electric circuit driven by an electrical battery. Nearly all living organisms derive their energy, directly or indirectly, from the radiant energy of sunlight, which arises from the thermonuclear fusion reactions that form helium in the sun (Fig. 1–8). Photosynthetic cells absorb the sun’s radiant energy and use it to drive electrons from water to carbon dioxide, forming energy-rich products such as starch and sucrose. In doing so, most photosynthetic organisms release molecular oxygen into the atmosphere. Ultimately, nonphotosynthetic organisms obtain energy for their needs by oxidizing the energy-rich products of photosynthesis, passing electrons to atmospheric oxygen to form water, carbon dioxide, and other end products, which are recycled in the environment. All of these reactions involving electron flow are oxidation-reduction reactions. Thus, other principles of the living state emerge:

The energy needs of virtually all organisms are provided, directly or indirectly, by solar energy.

The flow of electrons in oxidation-reduction reactions underlies energy transduction and energy conservation in living cells.

All living organisms are dependent on each other through exchanges of energy and matter via the environment.

Enzymes Promote Sequences of Chemical Reactions

The fact that a reaction is exergonic does not mean that it will necessarily proceed rapidly. The reaction coordinate diagram in Figure 1–6 (bottom) is actually an oversimplification. The path from reactant to product almost invariably involves an energy barrier, called the activation barrier (Fig. 1–9), that must be surmounted for any reaction to occur. The breaking and joining of bonds generally requires the prior bending or stretching of existing bonds, creating a transition state of higher free energy than either reactant or product. The highest point in the reaction coordinate diagram represents the transition state.

Activation barriers are crucial to the stability of biomolecules in living systems. Although, when isolated from other cellular components, most biomolecules are stable for days or even years, inside cells they often undergo chemical transformations within milliseconds. Without activation barriers, biomolecules within cells would rapidly break down to simple, low-energy forms. The lifetime of complex molecules would be very short, and the extraordinary continuity and organization of life would be impossible.

Virtually every cellular chemical reaction occurs because of enzymes – catalysts that are capable of greatly enhancing the rate of specific chemical reactions without being consumed in the process (Fig. 1–10). Enzymes, as catalysts, act by lowering this energy barrier between reactant and product. The activation energyG; Fig. 1–9) required to overcome this energy barrier could in principle be supplied by heating the reaction mixture, but this option is not available in living cells. Instead, during a reaction, enzymes bind reactant molecules in the transition state, thereby lowering the activation energy and enormously accelerating the rate of the reaction. The relationship between the activation energy and reaction rate is exponential; a small decrease in ΔG results in a very large increase in reaction rate. Enzyme-catalyzed reactions commonly proceed at rates up to 1010- to 1014-fold greater than the uncatalyzed rates.

Enzymes are, with a few exceptions we will consider later, proteins. Each enzyme protein is specific for the catalysis of a specific reaction, and each reaction in a cell is catalyzed by a different enzyme. Thousands of different types of enzymes are therefore required by each cell. The multiplicity of enzymes, their high specificity for reactants, and their susceptibility to regulation give cells the capacity to lower activation barriers selectively. This selectivity is crucial in the effective regulation of cellular processes.

The thousands of enzyme-catalyzed chemical reactions in cells are functionally organized into many different sequences of consecutive reactions called pathways, in which the product of one reaction becomes the reactant in the next (Fig. 1–11). Some of these sequences of enzyme-catalyzed reactions degrade organic nutrients into simple end products, in order to extract chemical energy and convert it into a form useful to the cell. Together these degradative, free-energy-yielding reactions are designated catabolism. Other enzyme-catalyzed pathways start from small precursor molecules and convert them to progressively larger and more complex molecules, including proteins and nucleic acids; such synthetic pathways invariably require the input of energy, and taken together represent anabolism. The network of enzyme-catalyzed pathways constitutes cellular metabolism.
 Figure 1–12 (a) Structural formula and (b) ball-and-stick model for adenosine triphosphate (ATP). The removal of the terminal phosphate of ATP is highly exergonic, and this reaction is coupled to many endergonic reactions in the cell.
 Figure 1–13 ATP is the chemical intermediate linking energy-releasing to energy-requiring cell processes. Its role in the cell is analogous to that of money in an economy: it is "earned/produced" in exergonic reactions and "spent/consumed" in endergonic ones.
 Figure 1–14 Regulation of a biosynthetic pathway by feedback inhibition. In the pathway by which isoleucine is formed in five steps from threonine (Fig. 1–11), the accumulation of the product isoleucine (F) causes inhibition of the first reaction in the pathway by binding to the enzyme catalyzing this reaction and reducing its activity. (The letters A to F represent the corresponding compounds shown in Fig. 1–11.)
ATP Is the Universal Carrier of Metabolic Energy, Linking Catabolism and Anabolism

Cells capture, store, and transport free energy in a chemical form. Adenosine triphosphate (ATP) (Fig. 1–12) functions as the major carrier of chemical energy in all cells. ATP carries energy between metabolic pathways by serving as the shared intermediate that couples endergonic reactions to exergonic ones. The terminal phosphate group of ATP is transferred to a variety of acceptor molecules, which are thereby activated for further chemical transformation. The adenosine diphosphate (ADP) that remains after the phosphate transfer is recycled to become ATP, at the expense of either chemical energy (during oxidative phosphorylation) or solar energy in photosynthetic cells (by the process of photophosphorylation). ATP is the major connecting link (the shared intermediate) between the catabolic and anabolic networks of enzyme-catalyzed reactions in the cell (Fig. 1–13).

These linked networks of enzyme-catalyzed reactions are virtually identical in all living organisms.

Metabolism Is Regulated to Achieve Balance and Economy

Not only can living cells simultaneously synthesize thousands of different kinds of carbohydrate, fat, protein, and nucleic acid molecules and their simpler subunits, they can also do so in the precise proportions required by the cell. For example, when rapid cell growth occurs, the precursors of proteins and nucleic acids must be made in large quantities, whereas in nongrowing cells the requirement for these precursors is much reduced. Key enzymes in each metabolic pathway are regulated so that each type of precursor molecule is produced in a quantity appropriate to the current requirements of the cell. Consider the pathway shown in Figure 1–14 (see also Fig. 1–11), which leads to the synthesis of isoleucine (one of the amino acids, the monomeric subunits of proteins). If a cell begins to produce more isoleucine than is needed for protein synthesis, the unused isoleucine accumulates. High concentrations of isoleucine inhibit the catalytic activity of the first enzyme in the pathway, immediately slowing the production of the amino acid. Such negative feedback keeps the production and utilization of each metabolic intermediate in balance.

Living cells also regulate the synthesis of their own catalysts, the enzymes. Thus a cell can switch off the synthesis of an enzyme required to make a given product whenever that product is available ready-made in the environment. These self-adjusting and self-regulating properties allow cells to maintain themselves in a dynamic steady state, despite fluctuations in the external environment.

Living cells are self-regulating chemical engines, adjusted for maximum economy.
Figure 1–15 Two ancient scripts. (a) The Prism of Sennacherib, inscribed in about 700 B.C., describes in characters of the Assyrian language some historical events during the reign of King Sennacherib. The Prism contains about 20,000 characters, weighs about 50 kg, and has survived almost intact for about 2,700 years. (b) The single DNA molecule of the bacterium E. coli, seen leaking out of a disrupted cell, is hundreds of times longer than the cell itself and contains all of the encoded information necessary to specify the cell’s structure and functions. The bacterial DNA contains about 10 million characters (nucleotides), weighs less than 10-10 g, and has undergone only relatively minor changes during the past several million years. The black spots and white specks are artifacts of the preparation.
 Figure 1–16 The complementary structure of double-stranded DNA accounts for its accurate replication. DNA is a linear polymer of four subunits, the deoxyribonucleotides deoxyadenylate (A), deoxyguanylate (G), deoxycytidylate (C), and deoxythymidylate (T), joined covalently. Each nucleotide has the intrinsic ability, due to its precise three-dimensional structure, to associate very specifically but noncovalently with one other nucleotide: A always associates with its complement T, and G with its complement C. In the doublestranded DNA molecule, the sequence of nucleotides in one strand is complementary to the sequence in the other; wherever G occurs in strand 1, C occurs in strand 2; wherever A occurs in strand 1, T occurs in strand 2. The two strands of the DNA, held together by a large number of hydrogen bonds (represented here by vertical blue lines) between the pairs of complementary nucleotides, twist about each other to form the DNA double helix. In DNA replication, prior to cell division, the two strands of the original DNA separate and two new strands are synthesized, each with a sequence complementary to one of the original strands. The result is two double-helical DNA molecules, each identical to the original DNA.
 Figure 1–17 The gradual accumulation of mutations over long periods of time results in new biological species, each with a unique DNA sequence. At top is shown a short segment of a gene in a hypothetical progenitor organism. With the passage of time, changes in nucleotide sequence (mutations, indicated here by colored boxes) occur, one at a time, resulting in progeny with different DNA sequences. These mutant progeny themselves undergo occasional mutations, yielding their own progeny differing by two or more nucleotides from the original sequence.
Biological Information Transfer

The continued existence of a biological species requires that its genetic information be maintained in a stable form and, at the same time, expressed with very few errors. Effective storage and accurate expression of the genetic message defines individual species, distinguishes them from one another, and assures their continuity over successive generations.

Among the seminal discoveries of twentieth-century biology are the chemical nature and the three-dimensional structure of the genetic material, DNA. The sequence of deoxyribonucleotides in this linear polymer encodes the instructions for forming all other cellular components and provides a template for the production of identical DNA molecules to be distributed to progeny when a cell divides.

Genetic Continuity Is Vested in DNA Molecules

Perhaps the most remarkable of all the properties of living cells and organisms is their ability to reproduce themselves with nearly perfect fidelity for countless generations. This continuity of inherited traits implies constancy, over thousands or millions of years, in the structure of the molecules that contain the genetic information. Very few historical records of civilization, even those etched in copper or carved in stone, have survived for a thousand years (Fig. 1–15). But there is good evidence that the genetic instructions in living organisms have remained nearly unchanged over very much longer periods; many bacteria have nearly the same size, shape, and internal structure and contain the same kinds of precursor molecules and enzymes as those that lived a billion years ago.

Hereditary information is preserved in DNA, a long, thin organic polymer so fragile that it will fragment from the shear forces arising in a solution that is stirred or pipetted. A human sperm or egg, carrying the accumulated hereditary information of millions of years of evolution, transmits these instructions in the form of DNA molecules, in which the linear sequence of covalently linked nucleotide subunits encodes the genetic message.
The Structure of DNA Allows for Its Repair and Replication with Near-Perfect Fidelity

The capacity of living cells to preserve their genetic material and to duplicate it for the next generation results from the structural complementarity between the two halves of the DNA molecule (Fig. 1–16). The basic unit of DNA is a linear polymer of four different monomeric subunits, deoxyribonucleotides (see Fig. 1–3), arranged in a precise linear sequence. It is this linear sequence that encodes the genetic information. Two of these polymeric strands are twisted about each other to form the DNA double helix, in which each monomeric subunit in one strand pairs specifically with the complementary subunit in the opposite strand. In the enzymatic replication or repair of DNA, one of the two strands serves as a template for the assembly of another, structurally complementary DNA strand. Before a cell divides, the two DNA strands separate and each serves as a template for the synthesis of a complementary strand, generating two identical double-helical molecules, one for each daughter cell. If one strand is damaged, continuity of information is assured by the information present on the other strand.

Genetic information is encoded in the linear sequence of four kinds of subunits of DNA.

The double-helical DNA molecule contains an internal template for its own replication and repair.

Changes in the Hereditary Instructions Allow Evolution

Despite the near-perfect fidelity of genetic replication, infrequent, unrepaired mistakes in the replication process produce changes in the nucleotide sequence of DNA, representing a genetic mutation (Fig. 1–17). Incorrectly repaired damage to one of the DNA strands has the same effect. Mutations can change the instructions for producing cellular components. Many mutations are deleterious or even lethal to the organism; they may, for example, cause the synthesis of a defective enzyme that is not able to catalyze an essential metabolic reaction.

Occasionally the mutation better equips an organism or cell to survive in its environment. The mutant enzyme might, for example, have acquired a slightly different specificity, so that it is now able to use as a reactant some compound that the cell was previously unable to metabolize. If a population of cells were to find itself in an environment where that compound was the only available source of fuel, the mutant cell would have an advantage over the other, unmutated (wild-type) cells in the population. The mutant cell and its progeny would survive in the new environment, whereas wild-type cells would starve and be eliminated.

Chance genetic variations in individuals in a population, combined with natural selection (survival of the fittest individuals in a challenging or changing environment), have resulted in the evolution of an enormous variety of organisms, each adapted to life in a particular ecological niche.
 Carolus Linnaeus
 Charles Darwin
 Figure 1–18 Linear sequences of deoxyribonucleotides in DNA, arranged into units known as genes, are transcribed into ribonucleic acid (RNA) molecules with complementary ribonucleotide sequences. The RNA sequences are then translated into linear protein chains, which fold spontaneously into their native three-dimensional shapes. Individual proteins sometimes associate with other proteins to form supramolecular complexes, stabilized by numerous weak interactions.
Molecular Anatomy Reveals Evolutionary Relationships

Biochemistry has confirmed and greatly extended evolutionary theory. Carolus Linnaeus recognized the anatomic similarities and differences among living organisms and provided a framework for assessing the relatedness of different species. Charles Darwin gave us a unifying hypothesis to explain the phylogeny of modern organisms – the origin of different species from a common ancestor. Biochemistry has begun to reveal the molecular anatomy of cells of different species – the sequences of subunits in nucleic acids and proteins and the three-dimensional structures of individual molecules of nucleic acid and protein. There is a reasonable prospect that when the twenty-first century dawns, we will know the entire nucleotide sequence of all of the genes that make up the biological heritage of a human.

At the molecular level, evolution is the emergence over time of different sequences of nucleotides within genes. With new genetic sequences being experimentally determined almost daily, biochemists have an enormously rich treasury of evidence with which to analyze evolutionary relationships and to refine evolutionary theory. The molecular phylogeny derived from gene sequences is consistent with, but in many cases more precise than, the classical phylogeny based on macroscopic structures.

Molecular structures and mechanisms have been conserved in evolution even though organisms have continuously diverged at the level of gross anatomy. At the molecular level, the basic unity of life is readily apparent; crucial molecular structures and mechanisms are remarkably similar from the simplest to the most complex organisms. Biochemistry makes it possible to discover the unifying features common to all life. This book examines many of these features: the mechanisms for energy conservation, biosynthesis, gene replication, and gene expression.

The Linear Sequence in DNA Encodes Proteins with Three-Dimensional Structures

The information in DNA is encoded as a linear (one-dimensional) sequence of the nucleotide units of DNA, but the expression of this information results in a three-dimensional cell. This change from one to three dimensions occurs in two phases. A linear sequence of deoxyribonucleotides in DNA codes (through the intermediary, RNA) for the production of a protein with a corresponding linear sequence of amino acids (Fig. 1–18). The protein folds itself into a particular three-dimensional shape, dictated by its amino acid sequence. The precise three-dimensional structure (native conformation) is crucial to the protein’s function as either catalyst or structural element. This principle emerges:

The linear sequence of amino acids in a protein leads to the acquisition of a unique three-dimensional structure by a self-assembly process.

Once a protein has folded into its native conformation, it may associate noncovalently with other proteins, or with nucleic acids or lipids,

to form supramolecular complexes such as chromosomes, ribosomes, and membranes (Fig. 1–18). These complexes are in many cases self-assembling. The individual molecules of these complexes have specific, high-affinity binding sites for each other, and within the cell they spontaneously form functional complexes.

Individual macromolecules with specific affinity for other macromolecules self-assemble into supramolecular complexes.

Noncovalent Interactions Stabilize Three-Dimensional Structures

The forces that provide stability and specificity to the three-dimensional structures of macromolecules and supramolecular complexes are mostly noncovalent interactions. These interactions, individually weak but collectively strong, include hydrogen bonds, ionic interactions among charged groups, van der Waals interactions, and hydrophobic interactions among nonpolar groups. These weak interactions are transient; individually they form and break in small fractions of a second. The transient nature of noncovalent interactions confers a flexibility on macromolecules that is critical to their function. Furthermore, the large number of noncovalent interactions in a single macromolecule makes it unlikely that at any given moment all the interactions will be broken; thus macromolecular structures are stable over time.

Three-dimensional biological structures combine the properties of flexibility and stability.

The flexibility and stability of the double-helical structure of DNA are due to the complementarity of its two strands and the many weak interactions between them. The flexibility of these interactions allows strand separation during DNA replication (see Fig. 1–16); the complementarity of the double helix is essential to genetic continuity.

Noncovalent interactions are also central to the specificity and catalytic efficiency of enzymes. Enzymes bind transition-state intermediates through numerous weak but precisely oriented interactions. Because the weak interactions are flexible, the complex survives the structural distortions as the reactant is converted into product.

The formation of noncovalent interactions provides the energy for self-assembly of macromolecules by stabilizing native conformations relative to unfolded, random forms. The native conformation of a protein is that in which the energetic advantages of forming weak interactions counterbalance the tendency of the protein chain to assume random forms. Given a specific linear sequence of amino acids and a specific set of conditions (temperature, ionic conditions, pH), a protein will assume its native conformation spontaneously, without a template or scaffold to direct the folding.

The Physical Roots of the Biochemical World

We can now summarize the various principles of the molecular logic of life:

A living cell is a self-contained, self-assembling, self-adjusting, self-perpetuating isothermal system of molecules that extracts free energy and raw materials from its environment.

The cell carries out many consecutive reactions promoted by specific catalysts, called enzymes, which it produces itself.

The cell maintains itself in a dynamic steady state, far from equilibrium with its surroundings. There is great economy of parts and processes, achieved by regulation of the catalytic activity of key enzymes.

Self-replication through many generations is ensured by the self-repairing, linear information-coding system. Genetic information encoded as sequences of nucleotide subunits in DNA and RNA specifies the sequence of amino acids in each distinct protein, which ultimately determines the three-dimensional structure and function of each protein.

Many weak (noncovalent) interactions, acting cooperatively, stabilize the three-dimensional structures of biomolecules and supramolecular complexes.

At no point in our examination of the molecular logic of living cells have we encountered any violation of known physical laws; nor have we needed to define new physical laws. The organic machinery of living cells functions within the same set of laws that governs the operation of inanimate machines, but the chemical reactions and regulatory processes of cells have been highly refined during evolution.

This set of principles has been most thoroughly validated in studies of unicellular organisms (such as the bacterium E. coli), which are exceptionally amenable to biochemical and genetic study. Although multicellular organisms must solve certain problems not encountered by unicellular organisms, such as the differentiation of the fertilized egg into specialized cell types, the same principles have been found to apply. Can such simple and mechanical statements apply to humans as well, with their extraordinary capacity for thought, language, and creativity? The pace of recent biochemical progress toward understanding such processes as gene regulation, cellular differentiation, communication among cells, and neural function has been extraordinarily fast, and is accelerating. The success of biochemical methods in solving and redefining these problems justifies the hope that the most complex functions of the most highly developed organism will eventually be explicable in molecular terms.

The relevant facts of biochemistry are many; the student approaching this subject for the first time may occasionally feel overwhelmed. Perhaps the most encouraging development in twentieth-

century biology is the realization that, for all of the enormous diversity in the biological world, there is a fundamental unity and simplicity to life. The organizing principles, the biochemical unity, and the evolutionary perspective of diversity, provided at the molecular level, will serve as helpful frames of reference for the study of biochemistry.
Further Reading

Asimov, I. (1962) Life and Energy: An Exploration of the Physical and Chemical Basis of Modern Biology, Doubleday & Co., Inc., New York.
An engaging account of the role of energy transformations in biology, written for the intelligent layman.

Blum, H.F. (1968) Time’s Arrow and Euolution, 3rd edn, Princeton University Press, Princeton, NJ.
An excellent discussion of the way the second law of thermodynamics has influenced biological euolution.

Dulbecco, R. (1987) The Design of Life, Yale University Press, New Haven, CT.
An unusual and excellent introduction to biology.

Fruton, J.S. (1972) Molecules and Life. Historical Essays on the Interplay of Chemistry and Biology, Wiley-Interscience, New York.
This series of essays describes the development of biochemistry from Pasteur’s studies of fermentation to the present studies of metabolism and information transfer. You may want to refer to these essays through this textbook.

Fruton, J.S. (1992) A Skeptical Biochemist, Harvard University Press, Cambridge, MA.

Hawking, S. (1988) A Brief History of Time, Bantam Books, Inc., New York.

Jacob, F. (1973) The Logic of Life: A History of Heredity, Pantheon Books, Inc., New York. Originally published (1970) as La logique du vivant: une histoire de l’hérédité, Editions Gallimard, Paris.
A fascinating historical and philosophical account of the route by which we came to the present molecular understanding of life.

Kornberg, A. (1987) The two cultures: chemistry and biology. Biochem. 26, 6888–6891.
The importance of applying chemical tools to biological problems, described by an eminent practitioner.

Monod, J. (1971) Chance and Necessity, Alfred A. Knopf, Inc., New York. [Paperback version (1972) Vintage Books, New York.] Originally published (1970) as Le hasard et la necessité, Editions du Seuil, Paris.
An exploration of the philosophical implications of biological knowledge.

Schrödinger, E. (1944) What is Life?, Cambridge University Press, New York. [Reprinted (1956) in What is Life? and Other Scientific Essays, Doubleday Anchor Books, Garden City, NY.]
A thought-provoking look at life, written by a prominent physical chemist.
 Figure 2–1 The universal features of all living cells: a nucleus or nucleoid, a plasma membrane, and cytoplasm.
The cytosol is that portion of the cytoplasm that remains in the supernatant after centrifugation of a cell extract of 150,000 g for 1 h.
Chapter 2
Cells are the structural and functional units of all living organisms. The smallest organisms consist of single cells and are microscopic, whereas larger organisms are multicellular. The human body, for example, contains at least 1014 cells. Unicellular organisms are found in great variety throughout virtually every environment from Antarctica to hot springs to the inner recesses of larger organisms. Multicellular organisms contain many different types of cells, which vary in size, shape, and specialized function. Yet no matter how large and complex the organism, each of its cells retains some individuality and independence.

Despite their many differences, cells of all kinds share certain structural features (Fig. 2–1). The plasma membrane defines the periphery of the cell, separating its contents from the surroundings. It is composed of enormous numbers of lipids and protein molecules, held together primarily by noncovalent hydrophobic interactions (p. 18), forming a thin, tough, pliable, hydrophobic bilayer around the cell. The membrane is a barrier to the free passage of inorganic ions and most other charged or polar compounds; transport proteins in the plasma membrane allow the passage of certain ions and molecules. Other membrane proteins are receptors that transmit signals from the outside to the inside of the cell, or are enzymes that participate in membrane-associated reaction pathways.

Because the individual lipid and protein subunits of the plasma membrane are not covalently linked, the entire structure is remarkably flexible, allowing changes in the shape and size of the cell. As a cell grows, newly made lipid and protein molecules are inserted into its plasma membrane; cell division produces two cells, each with its own membrane. Growth and fission occur without loss of membrane integrity. In a reversal of the fission process, two separate membrane surfaces can fuse, also without loss of integrity. Membrane fusion and fission are central to mechanisms of transport known as endocytosis and exocytosis.

The internal volume bounded by the plasma membrane, the cytoplasm, is composed of an aqueous solution, the cytosol, and a variety of insoluble, suspended particles (Fig. 2–1). The cytosol is not simply a dilute aqueous solution; it has a complex composition and gel-like consistency. Dissolved in the cytosol are many enzymes and the RNA molecules that encode them; the monomeric subunits (amino acids and nucleotides) from which these macromolecules are assembled; hundreds of small organic molecules called metabolites, intermediates in biosynthetic and degradative pathways; coenzymes, compounds of

intermediate molecular weight (Mr 200 to 1,000) that are essential participants in many enzyme-catalyzed reactions; and inorganic ions.

Among the particles suspended in the cytosol are supramolecular complexes and, in higher organisms but not in bacteria, a variety of membrane-bounded organelles in which specialized metabolic machinery is localized. Ribosomes, complexes of over 50 different protein and RNA molecules, are small particles, 18 to 22 nm in diameter. Ribosomes are the enzymatic machines on which protein synthesis occurs; they often occur in clusters called polysomes (polyribosomes) held together by a strand of messenger RNA. Also present in the cytoplasm of many cells are granules containing stored nutrients such as starch and fat. Nearly all living cells have either a nucleus or a nucleoid, in which the genome (the complete set of genes, composed of DNA) is stored and replicated. The DNA molecules are always very much longer than the cells themselves, and are tightly folded and packed within the nucleus or nucleoid as supramolecular complexes of DNA with specific proteins. The bacterial nucleoid is not separated from the cytoplasm by a membrane, but in higher organisms, the nuclear material is enclosed within a double membrane, the nuclear envelope. Cells with nuclear envelopes are called eukaryotes (Greek eu, "true," and karyon, "nucleus"); those without nuclear envelopes – bacterial cells – are prokaryotes (Greek pro, "before"). Unlike bacteria, eukaryotes have a variety of other membrane-bounded organelles in their cytoplasm, including mitochondria, lysosomes, endoplasmic reticulum, Golgi complexes, and, in photosynthetic cells, chloroplasts.

In this chapter we review briefly the evolutionary relationships among some commonly studied cells and organisms, and the structural features that distinguish cells of various types. Our main focus is on eukaryotic cells. Also discussed in brief are the cellular parasites known as viruses.

 Figure 2–2 Smaller cells have larger ratios of surface area to volume, and their interiors are therefore more accessible to substances diffusing into the cell through the surface. When the large cube (representing a large cell) is subdivided into many smaller cubes (cells), the total surface area increases greatly without a change in the total volume, and the surface-to-volume ratio increases accordingly.
 Figure 2–3 Convolutions of the plasma membrane, or long, thin extensions of the cytoplasm, increase the surface-to-volume ratio of cells. (a) Cells of the intestinal mucosa (the inner lining of the small intestine) are covered with microvilli, increasing the area for absorption of nutrients from the intestine. (b) Neurons of the hippocampus of the rat brain are several millimeters long, but the long extensions (axons) are only about 10 nm wide.
Cellular Dimensions

Most cells are of microscopic size. Animal and plant cells are typically 10 to 30 μm in diameter, and many bacteria are only 1 to 2 μm long.

What limits the dimensions of a cell? The lower limit is probably set by the minimum number of each of the different biomolecules required by the cell. The smallest complete cells, certain bacteria known collectively as mycoplasma, are 300 nm in diameter and have a volume of about 10-14 mL. A single ribosome is about 20 nm in its longest dimension, so a few ribosomes take up a substantial fraction of the cell’s volume. In a cell of this size, a 1 μM solution of a compound represents only 6,000 molecules.

The upper limit of cell size is set by the rate of diffusion of solute molecules in aqueous systems. The availability of fuels and essential nutrients from the surrounding medium is sometimes limited by the rate of their diffusion to all regions of the cell. A bacterial cell that depends upon oxygen-consuming reactions for energy production (an aerobic cell) must obtain molecular oxygen (O2) from the surrounding medium by diffusion through its plasma membrane. The cell is so small, and the ratio of its surface area to its volume is so large, that every part of its cytoplasm is easily reached by O2 diffusing into the cell. As the size of a cell increases, its surface-to-volume ratio decreases (Fig. 2–2), until metabolism consumes O2 faster than diffusion can

supply it. Aerobic metabolism thus becomes impossible as cell size increases beyond a certain point, placing a theoretical upper limit on the size of the aerobic cell.

There are interesting exceptions to this generalization that cells must be small. The giant alga Nitella has cells several centimeters long. To assure the delivery of nutrients, metabolites, and genetic information (RNA) to all of its parts, each cell is vigorously "stirred" by active cytoplasmic streaming (p. 43). The shape of a cell can also help to compensate for its large size. A smooth sphere has the smallest surface-to-volume ratio possible for a given volume. Many large cells, although roughly spherical, have highly convoluted surfaces (Fig. 2–3a), creating larger surface areas for the same volume and thus facilitating the uptake of fuels and nutrients and release of waste products to the surrounding medium. Other large cells (neurons, for example) have large surface-to-volume ratios because they are long and thin, star-shaped, or highly branched (Fig. 2–3b), rather than spherical.

Usefulness of Cells and Organisms in Biochemical Studies

Because all living cells have evolved from the same progenitors, they share certain fundamental similarities. Careful biochemical study of just a few cells, however different in biochemical details and varied in superficial appearance, ought to yield general principles applicable to all cells and organisms. The burgeoning knowledge in biology in the past 150 years has supported these propositions over and over again. Certain cells, tissues, and organisms have proved more amenable to experimental studies than others. Knowledge in biochemistry, and much of the information in this book, continues to be derived from a few representative tissues and organisms, such as the bacterium Escherichia coli, the yeast Saccharomyces, photosynthetic algae, spinach leaves, the rat liver, and the skeletal muscle of several different vertebrates.

In the isolation of enzymes and other cellular components, it is ideal if the experimenter can begin with a plentiful and homogeneous source of the material. The component of interest (such as an enzyme or nucleic acid) often represents only a miniscule fraction of the total material, and many grams of starting material are needed to obtain a few micrograms of the purified component. Certain types of physical and chemical studies of biomolecules are precluded if only microgram quantities of the pure substance are available. A homogeneous source of an enzyme or nucleic acid, in which all of the cells are genetically and biochemically identical, leaves no doubt about which cell type yielded the purified component, and makes it safer to extrapolate the results of in vitro studies to the situation in vivo. A large culture of bacterial or protistan cells (E. coli, Saccharomyces, or Chlamydomonas, for example), all derived by division from the same parent and therefore genetically identical, meets the requirement for a plentiful and homogeneous source. Individual tissues from laboratory animals (rat liver, pig brain, rabbit muscle) are plentiful sources of similar, though not identical; cells. Some animal and plant cells proliferate in cell culture, producing populations of identical (cloned) cells in quantities suitable for biochemical analysis.

Genetic mutants, in which a defect in a single gene produces a specific functional defect in the cell or organism, are extremely useful in establishing that a certain cellular component is essential to a particular cellular function. Because it is technically much simpler to produce and detect mutants in bacteria and yeast, these organisms (E. coli and Saccharomyces cerevisiae, for example) have been favorite experimental targets for biochemical geneticists.

An organism that is easy to culture in the laboratory, with a short generation time, offers significant advantages to the research biochemist. An organism that requires only a few simple precursor molecules in its growth medium can be cultured in the presence of a radioisotopically labeled precursor, and the metabolic fate of that precursor can then be conveniently traced by following the incorporation of the radioactive atoms into its metabolic products. The short generation time (minutes or hours) of microorganisms allows the investigator to follow a labeled precursor or a genetic defect through many generations in a few days. In higher organisms with generation times of months or years, this is virtually impossible.

Some highly specialized tissues of multicellular organisms are

remarkably enriched in some particular component related to their specialized function. Vertebrate skeletal muscle is a rich source of actin and myosin; pancreatic secretory cells contain high concentrations of rough endoplasmic reticulum; sperm cells are rich in DNA and in flagellar proteins; liver (the major biosynthetic organ of vertebrates) contains high concentrations of many enzymes of biosynthetic pathways; spinach leaves contain large numbers of chloroplasts; and so on. For studies on such specific components or processes, biochemists commonly choose a specialized tissue for their experimental systems.

Sometimes simplicity of structure or function makes a particular cell or organism attractive as an experimental system. For studies of plasma membrane structure and function, the mature erythrocyte (red blood cell) has been a favorite; it has no internal membranes to complicate purification of the plasma membrane. Some bacterial viruses (bacteriophages) have few genes. Their DNA molecules are therefore smaller and much simpler than those of humans or corn plants, and it has proved easier to study replication in these viruses than in human or corn chromosomes.

The biochemical description of living cells in this book is a composite, based on studies of many types of cells. The biochemist must always exercise caution in generalizing from results obtained in studies of selected cells, tissues, and organisms, and in relating what is observed in vitro to what happens within the living cell.

 Figure 2–4 Organisms can be classified according to their source of energy (shaded red) and the form in which they obtain carbon atoms (shaded blue) for the synthesis of cellular material. Organic compounds are both energy source and carbon source for chemoheterotrophs such as ourselves. Some, but not all, chemoheterotrophs consume O2 and produce CO2, and some photoautotrophs produce O2 (shaded green).

Evolution and Structure of Prokaryotic Cells

All of the organisms alive today are believed to have evolved from ancient, unicellular progenitors. Two large groups of extant prokaryotes evolved from these early forms: archaebacteria (Greek, arché, "origin") and eubacteria. Eubacteria inhabit the soil, surface waters, and the tissues of other living or decaying organisms. Most common and well-studied bacteria, including E. coli and the cyanobacteria (formerly called blue-green algae), are eubacteria. The archaebacteria are more recently discovered and less well studied. They inhabit more extreme environments – salt brines, hot acid springs, bogs, and the deep regions of the ocean.

Within each of these two large groups of bacteria are subgroups distinguished by the habitats to which they are best adapted. In some habitats there is a plentiful supply of oxygen, and the resident organisms live by aerobic metabolism; their catabolic processes ultimately result in the transfer of electrons from fuel molecules to oxygen. Other environments are virtually devoid of oxygen, forcing resident organisms to conduct their catabolic business without it. Many of the organisms that have evolved in these anaerobic environments are obligate anaerobes; they die when exposed to oxygen.

All organisms, including bacteria, can be classified as either chemotrophs (those obtaining their energy from a chemical fuel) or phototrophs (those using sunlight as their primary energy source). Certain organisms can synthesize some or all of their monomeric subunits, metabolic intermediates, and macromolecules from very simple starting materials such as CO2 and NH3; these are the autotrophs. Others must acquire some of their nutrients from the environment preformed (by autotrophic organisms, for example); these are heterotrophs. There are therefore four general modes of obtaining fuel and energy, and four general groups of organisms distinguished by these

modes: chemoheterotrophs, chemoautotrophs, photoheterotrophs, and photoautotrophs (Fig. 2–4).

As shown in Figure 2–5, the earliest cells probably arose about 3.5 billion (3.5 x 109) years ago in the rich mixture of organic compounds, the "primordial soup", of prebiotic times; they were almost certainly chemoheterotrophs. The organic compounds were originally synthesized from such components of the early earth’s atmosphere as CO, CO2, N2, and CH4 by the nonbiological actions of volcanic heat and lightning (Chapter 3). Primitive heterotrophs gradually acquired the capability to derive energy from certain compounds in their environment and to use that energy to synthesize more and more of their own precursor molecules, thereby becoming less dependent on outside sources of these molecules – less extremely heterotrophic. A very significant evolutionary event was the development of pigments capable of capturing visible light from the sun and using the energy to reduce or "fix" CO2 into more complex organic compounds. The original electron (hydrogen) donor for these photosynthetic organisms was probably H2S, yielding elemental sulfur as the byproduct, but at some point cells developed the enzymatic capacity to use H2O as the electron donor in photosynthetic reactions, producing O2. The cyanobacteria are the modern descendants of these early photosynthetic O2 producers.

The atmosphere of the earth in the earliest stages of biological evolution was nearly devoid of O2, and the earliest cells were therefore anaerobic. With the rise of O2-producing photosynthetic cells, the earth’s atmosphere became progressively richer in O2, allowing the evolution of aerobic organisms, which obtained energy by passing electrons from fuel molecules to O2 (that is, by oxidizing organic compounds). Because electron transfers involving O2 yield energy (they are very exergonic; see Chapter 1), aerobic organisms enjoyed an energetic advantage over their anaerobic counterparts when both competed in an environment containing O2. This advantage translated into the predominance of aerobic organisms in O2-rich environments.

Modern bacteria inhabit almost every ecological niche in the biosphere, and there are bacterial species capable of using virtually every type of organic compound as a source of carbon and energy. Perhaps three-fourths of all the living matter on the earth consists of microscopic organisms, most of them bacteria.

Figure 2–5  Landmarks in the evolution of life on earth.

Bacteria play an important role in the biological exchanges of matter and energy. Photosynthetic bacteria in both fresh and marine waters trap solar energy and use it to generate carbohydrates and other cell materials, which are in turn used as food by other forms of life. Some bacteria can capture molecular nitrogen (N2) from the atmosphere and use it to form biologically useful nitrogenous compounds, a process known as nitrogen fixation. Because animals and most plants cannot do this, bacteria form the starting point of many food chains in the biosphere. They also participate as ultimate consumers, degrading the organic structures of dead plants and animals and recycling the end products to the environment.
Figure 2–6  Common structural features of bacterial cells. Because of differences in cell envelope structure, some eubacteria (gram-positive bacteria) retain Gram’s stain, and others (gram-negative bacteria) do not. E. coli is gram-negative. Cyanobacteria are also eubacteria, but are distinguished by their extensive internal membrane system, in which photosynthetic pigments are localized.
Escherichia coli  Is the Best-Studied Prokaryotic Cell

Bacterial cells share certain common structural features, but also show group-specific specializations (Fig. 2–6). E. coli is a usually harmless inhabitant of the intestinal tract of human beings and many other mammals. The E. coli cell is about 2 μm long and a little less than 1 μm in diameter. It has a protective outer membrane and an inner plasma membrane that encloses the cytoplasm and the nucleoid. Between the inner and outer membranes is a thin but strong layer of peptidoglycans (sugar polymers cross-linked by amino acids), which gives the cell its shape and rigidity. The plasma membrane and the layers outside it constitute the cell envelope. Differences in the cell envelope account for the different affinities for the dye Gentian violet, which is the basis for Gram’s stain; gram-positive bacteria retain the dye, and gram-negative bacteria do not. The outer membrane of E. coli, like that of other gram-negative eubacteria, is similar to the plasma membrane in structure but is different in composition. In gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus, for example) there is no outer membrane, and the peptidoglycan layer surrounding the plasma membrane is much thicker than that in gram-negative bacteria. The plasma membranes of eubacteria consist of a thin bilayer of lipid molecules penetrated by proteins. Archaebacterial membranes have a similar architecture, although their lipids differ from those of the eubacteria.

The plasma membrane contains proteins capable of transporting certain ions and compounds into the cell and carrying products and waste out. Also in the plasma membrane of most eubacteria are electron-carrying proteins (cytochromes) essential in the formation of ATP from ADP (Chapter 1). In the photosynthetic bacteria, internal membranes derived from the plasma membrane contain chlorophyll and other light-trapping pigments.

From the outer membrane of E. coli cells and some other eubacteria protrude short, hairlike structures called pili, by which cells adhere to the surfaces of other cells. Strains of E. coli and other motile bacteria have one or more long flagella, which can propel the bacterium through its aqueous surroundings. Bacterial flagella are thin, rigid, helical rods, 10 to 20 nm thick. They are attached to a protein structure that spins in the plane of the cell surface, rotating the flagellum.

The cytoplasm of E. coli contains about 15,000 ribosomes, thousands of copies of each of several thousand different enzymes, numerous metabolites and cofactors, and a variety of inorganic ions. Under some conditions, granules of polysaccharides or droplets of lipid accumulate. The nucleoid contains a single, circular molecule of DNA. Although the DNA molecule of an E. coli cell is almost 1,000 times longer

than the cell itself, it is packaged with proteins and tightly folded into the nucleoid, which is less than 1 μm in its longest dimension. As in all bacteria, no membrane surrounds the genetic material. In addition to the DNA in the nucleoid, the cytoplasm of most bacteria contains many smaller, circular segments of DNA called plasmids. These nonessential segments of DNA are especially amenable to experimental manipulation and are extremely useful to the molecular geneticist. In nature, some plasmids confer resistance to toxins and antibiotics in the environment.

There is a primitive division of labor within the bacterial cell. The cell envelope regulates the flow of materials into and out of the cell, and protects the cell from noxious environmental agents. The plasma membrane and the cytoplasm contain a variety of enzymes essential to energy metabolism and the synthesis of precursor molecules; the ribosomes manufacture proteins; and the nucleoid stores and transmits genetic information. Most bacteria lead existences that are nearly independent of other cells, but some bacterial species tend to associate in clusters or filaments, and a few (the myxobacteria, for example) demonstrate primitive social behavior. Only eukaryotic cells, however, form true multicellular organisms with a division of labor among cell types.

Evolution of Eukaryotic Cells

Fossils older than 1.5 billion years are limited to those from small and relatively simple organisms, similar in size and shape to modern prokaryotes. Starting about 1.5 billion years ago, the fossil record begins to show evidence of larger and more complex organisms, probably the earliest eukaryotic cells (see Fig. 2–5). Details of the evolutionary path from prokaryotes to eukaryotes cannot be deduced from the fossil record alone, but morphological and biochemical comparison of modern organisms has suggested a reasonable sequence of events consistent with the fossil evidence.

Figure 2–7 One view of how modern plants, animals, fungi, protists, and bacteria share a common evolutionary precursor.
Eukaryotic Cells Evolved from Prokaryotes in Several Stages

Three major changes must have occurred as prokaryotes gave rise to eukaryotes (Fig. 2–7). First, as cells acquired more DNA (Table 2–1), mechanisms evolved to fold it compactly into discrete complexes with specific proteins and to divide it equally between daughter cells at cell division. These DNA-protein complexes, chromosomes, (Greek chroma, "color" and soma, "body"), become especially compact at the time of cell division, when they can be visualized with the light microscope as threads of chromatin. Second, as cells became larger, a system of intracellular membranes developed, including a double membrane surrounding the DNA. This membrane segregated the nuclear process of RNA synthesis using a DNA template from the cytoplasmic process of protein synthesis on ribosomes. Finally, primitive eukaryotic cells, which were incapable of photosynthesis or of aerobic metabolism, pooled their assets with those of aerobic bacteria or photosynthetic bacteria to form symbiotic associations that became permanent. Some aerobic bacteria evolved into the mitochondria of modern eukaryotes, and some photosynthetic cyanobacteria became the chloroplasts of modern plant cells. Prokaryotic and eukaryotic cells are compared in Table 2–2.

Early Eukaryotic Cells Gave Rise to Diverse Protists

With the rise of primitive eukaryotic cells, further evolution led to a tremendous diversity of unicellular eukaryotic organisms (protists). Some of these (those with chloroplasts) resembled modern photosynthetic protists such as Euglena and Chlamydomonas; other, nonphotosynthetic protists were more like Paramecium or Dictyostelium. Unicellular eukaryotes are abundant, and the cells of all multicellular animals, plants, and fungi are eukaryotic; there are only a few thousand prokaryotic species, but millions of species of eukaryotic organisms.

Figure 2–8 Schematic illustration of the two types of eukaryotic cell: a representative animal cell (a) and a representative plant cell (b).
Major Structural Features of Eukaryotic Cells

Typical eukaryotic cells (Fig. 2–8) are much larger than prokaryotic cells – commonly 10 to 30 μm in diameter, with cell volumes 1,000 to 10,000 times larger than those of bacteria. The distinguishing characteristic of eukaryotes is the nucleus with a complex internal structure, surrounded by a double membrane. The other striking difference between eukaryotes and prokaryotes is that eukaryotes contain a number of other membrane-bounded organelles. The following sections describe the structures and roles of the components of eukaryotic cells in more detail.

Figure 2–9 Proteins in the plasma membrane serve as transporters, signal receptors, and ion channels. Extracellular signals are amplified by receptors, because binding of a single ligand molecule to the surface receptor causes many molecules of an intracellular signal molecule to be formed, or many ions to flow through the opened channel. Transporters carry substances into and out of the cell, but do not act as signal amplifiers.
The Plasma Membrane Contains Transporters and Receptors

The external surface of a cell is in contact with other cells, the extracellular fluid, and the solutes, nutrient molecules, hormones, neurotransmitters, and antigens in that fluid. The plasma membranes of all cells contain a variety of transporters, proteins that span the width of the membrane and carry nutrients into and waste products out of the cell. Cells also have surface membrane proteins (signal receptors) that present highly specific binding sites for extracellular signaling molecules (receptor ligands). When an external ligand binds to its specific receptor, the receptor protein transduces the signal carried by that ligand into an intracellular message (Fig. 2–9). For example, some surface receptors are associated with ion channels that open when the receptor is occupied; others span the membrane and activate or inhibit cellular enzymes on the inner membrane surface. Whatever the mode of signal transduction, surface receptors characteristically act as signal amplifiers – a single ligand molecule bound to a single receptor may cause the flux of thousands of ions through an opened channel, or the synthesis of thousands of molecules of an intracellular messenger molecule by an activated enzyme.

Some surface receptors recognize ligands of low molecular weight, and others recognize macromolecules. For example, binding of acetylcholine (Mr 146) to its receptor begins a cascade of cellular events that underlie the transmission of signals for muscle contraction. Blood proteins (Mr > 20,000) that carry lipids (lipoproteins) are recognized by specific cell surface receptors and then transported into the cells. Antigens (proteins, viruses, or bacteria, recognized by the immune system as foreign) bind to specific receptors and trigger the production of antibodies. During the development of multicellular organisms, neighboring cells influence each other’s developmental paths, as signal molecules from one cell type react with receptors of other cells. Thus the surface membrane of a cell is a complex mosaic of different kinds of highly specific "molecular antennae" through which cells receive, amplify, and react to external signals.

Most cells of higher plants have a cell wall outside the plasma membrane (Fig. 2–8b), which serves as a rigid, protective shell. The cell wall, composed of cellulose and other carbohydrate polymers, is thick but porous. It allows water and small molecules to pass readily, but swelling of the cell due to the accumulation of water is resisted by the rigidity of the wall.
Endocytosis and Exocytosis Carry Traffic across the Plasma Membrane

Endocytosis is a mechanism for transporting components of the surrounding medium deep into the cytoplasm. In this process (Fig. 2–10), a region of the plasma membrane invaginates, enclosing a small volume of extracellular fluid within a bud that pinches off inside the cell by membrane fission. The resulting small vesicle (endosome) can move into the interior of the cell, delivering its contents to another organelle bounded by a single membrane (a lysosome, for example; see p. 34) by fusion of the two membranes. The endosome thus serves as an intracellular extension of the plasma membrane, effectively allowing intimate contact between components of the extracellular medium and regions deep within the cytoplasm, which could not be reached by diffusion alone. Phagocytosis is a special case of endocytosis, in which the material carried into the cell (within a phagosome) is particulate, such as a cell fragment or even another, smaller cell. The inverse of endocytosis is exocytosis (Fig. 2–10), in which a vesicle in the cytoplasm moves to the inside surface of the plasma membrane and fuses with it, releasing the vesicular contents outside the membrane. Many proteins destined for secretion into the extracellular space are released by exocytosis after being packaged into secretory vesicles.

Figure 2–10  The endomembrane system includes the nuclear envelope, endoplasmic reticulum, Golgi complex, and several types of small vesicles. This system encloses a compartment (the lumen) distinct from
The Endoplasmic Reticulum Organizes the Synthesis of Proteins and Lipids

The small transport vesicles moving to and from the plasma membrane in exocytosis and endocytosis are parts of a dynamic system of intracellular membranes (Fig. 2–10), which includes the endoplasmic reticulum, the Golgi complexes, the nuclear envelope, and a variety of small vesicles such as lysosomes and peroxisomes. Although generally represented as discrete and static elements, these structures are in fact in constant flux, with membrane vesicles continually budding from one of the structures and moving to and merging with another.

The endoplasmic reticulum is a highly convoluted, three-dimensional network of membrane-enclosed spaces extending throughout the cytoplasm and enclosing a subcellular compartment (the lumen of the endoplasmic reticulum) separate from the cytoplasm. The many flattened branches (cisternae) of this compartment are continuous with each other and with the nuclear envelope. In cells specialized for the secretion of proteins into the extracellular space, such as the pancreatic cells that secrete the hormone insulin, the endoplasmic reticulum is particularly prominent. The ribosomes that synthesize proteins destined for export attach to the outer (cytoplasmic) surface of the endoplasmic reticulum, and the secretory proteins are passed through the membrane into the lumen as they are synthesized. Proteins destined for sequestration within lysosomes, or for insertion into the nuclear or plasma membranes, are also synthesized on ribosomes attached to the endoplasmic reticulum. By contrast, proteins that will remain and function within the cytosol are synthesized on cytoplasmic ribosomes unassociated with the endoplasmic reticulum.

The attachment of thousands of ribosomes (usually in regions of large cisternae) gives the rough endoplasmic reticulum its granular appearance (Fig. 2–10) and thus its name. In other regions of the cell, the endoplasmic reticulum is free of ribosomes. This smooth endoplasmic reticulum, which is physically continuous with the rough

endoplasmic reticulum, is the site of lipid biosynthesis and of a variety of other important processes, including the metabolism of certain drugs and toxic compounds. Smooth endoplasmic reticulum is generally tubular, in contrast to the long, flattened cisternae typical of rough endoplasmic reticulum. In some tissues (skeletal muscle, for example) the endoplasmic reticulum is specialized for the storage and rapid release of calcium ions. Ca2+ release is the trigger for many cellular events, including muscle contraction.
the cytosol. Contents of the lumen move from one region of the endomembrane system to another as small transport vesicles bud from one component and fuse with another. High-magnification electron micrographs of a sectioned cell show rough endoplasmic reticulum, studded with ribosomes, smooth endoplasmic reticulum, and the Golgi complex.

The endomembrane system is dynamic; newly synthesized proteins move into the lumen of the rough endoplasmic reticulum and thus to the smooth endoplasmic reticulum, then to the Golgi complex via transport vesicles. In the Golgi complex, molecular "addresses" are added to specific proteins to direct them to the cell surface, lysosomes, or secretory vesicles. The contents of secretory vesicles are released from the cell by exocytosis. Endocytosis and phagocytosis bring extracellular materials into the cell. Fusion of endosomes (or phagosomes) with lysosomes, which are full of digestive enzymes, results in the degradation of the extracellular materials.

The Golgi Complex Processes and Sorts Protein

Nearly all eukaryotic cells have characteristic clusters of membrane vesicles called dictyosomes. Several connected dictyosomes constitute a Golgi complex. A Golgi complex (also called Golgi apparatus) is most commonly seen as a stack of flattened membrane vesicles (cisternae) (Fig. 2–10). Near the ends of these cisternae are numerous, much smaller, spherical vesicles (transport vesicles) that bud off the edges of the cisternae.

The Golgi complex is asymmetric, structurally and functionally. The cis side faces the rough endoplasmic reticulum, and the trans side, the plasma membrane; between these are the medial elements. Proteins, during their synthesis on ribosomes bound to the rough endoplasmic reticulum, are inserted into the interior (lumen) of the cisternae. Small membrane vesicles containing the newly synthesized proteins bud from the endoplasmic reticulum and move to the Golgi complex, fusing with the cis side. As the proteins pass through the Golgi complex to the trans side, enzymes in the complex modify the protein molecules by adding sulfate, carbohydrate, or lipid moieties to side chains of certain amino acids. One of the functions of this modification of a newly synthesized protein is to "address" it to its proper destination as it leaves the Golgi complex in a transport vesicle budding from the trans side. Certain proteins are enclosed in secretory vesicles, eventually to be released from the cell by exocytosis. Others are targeted for intracellular organelles such as lysosomes, or for incorporation into the plasma membrane during cell growth.

Lysosomes Are Packets of Hydrolyzing Enzymes

Lysosomes, found in the cytoplasm of animal cells, are spherical vesicles bounded by a single membrane. They are usually about 1 μm in diameter, about the size of a small bacterium (Fig. 2–10). Lysosomes contain enzymes capable of digesting proteins, polysaccharides, nucleic acids, and lipids. They function as cellular recycling centers for complex molecules brought into the cell by endocytosis, fragments of foreign cells brought in by phagocytosis, or worn-out organelles from the cell’s own cytoplasm. These materials selectively enter the lysosomes by fusion of the lysosomal membrane with endosomes, phagosomes, or defective organelles, and are then degraded to their simple components (amino acids, monosaccharides, fatty acids, etc.), which are released into the cytosol to be recycled into new cellular components or further catabolized.

The degradative enzymes within lysosomes would be harmful if not confined by the lysosomal membrane; they would be free to act on all cellular components. The lysosomal compartment is more acidic (pH ≤ 5) than the cytoplasm (pH ≈ 7); the acidity is due to the action of an ATP-fueled proton pump in the lysosomal membrane. Lysosomal enzymes are much less active at pH 7 than at pH ≤ 5, which provides a second line of defense against destruction of cytosolic macromolecules, should these enzymes escape into the cytosol.

Figure 2–11  The vacuole of a plant cell contains high concentrations of a variety of stored compounds and waste products. Water enters the vacuole by osmosis and increases the vacuolar volume. The resulting turgor pressure forces the cytoplasm out against the cell wall. The rigidity of the cell wall prevents expansion and rupture of the plasma membrane.
Vacuoles of Plant Cells Play Several Important Roles

Plant cells do not have organelles identical to lysosomes, but their vacuoles carry out similar degradative reactions as well as other functions

not found in animal cells. Growing plant cells contain several small vacuoles, vesicles bounded by a single membrane, which fuse and become one large vacuole in the center of the mature cell (Fig. 2–11; see also Fig. 2–8b). The surrounding membrane, the tonoplast, regulates the entry into the vacuole of ions, metabolites, and cellular structures destined for degradation. In the mature cell, the vacuole may represent as much as 90% of the total cell volume, pressing the cytoplasm into a thin layer between the tonoplast and the plasma membrane. The liquid within the vacuole, the cell sap, contains digestive enzymes that degrade and recycle macromolecular components no longer useful to the cell. In some plant cells, the vacuole contains high concentrations of pigments (anthocyanins) that give the deep purple and red colors to the flowers of roses and geraniums and the fruits of grapes and plums. Like the contents of lysosomes, the cell sap is generally more acidic than the surrounding cytosol. In addition to its role in storage and degradation of cellular components, the vacuole also provides physical support to the plant cell. Water passes into the vacuole by osmosis because of the high solute concentration of the cell sap, creating outward pressure on the cytosol and the cell wall. This turgor pressure within cells stiffens the plant tissue (Fig. 2–11).
Peroxisomes Destroy Hydrogen Peroxide, and Glyoxysomes Convert Fats to Carbohydrates

Some of the oxidative reactions in the breakdown of amino acids and fats produce free radicals and hydrogen peroxide (H2O2), very reactive chemical species that could damage cellular machinery. To protect the cell from these destructive byproducts, such reactions are segregated within small membrane-bounded vesicles called peroxisomes. The hydrogen peroxide is degraded by catalase, an enzyme present in large quantities in peroxisomes and glyoxysomes; it catalyzes the reaction 2H2O2 → 2H2O + O2.

Glyoxysomes are specialized peroxisomes found in certain plan cells. They contain high concentrations of the enzymes of the glyoxylate cycle, a metabolic pathway unique to plants that allows the conversion of stored fats into carbohydrates during seed germination. Lysosomes, peroxisomes, and glyoxysomes are sometimes referred to collectively as microbodies.
Figure 2–12 The nucleus and nuclear envelope.
(a) Scanning electron micrograph of the surface of the nuclear envelope, showing numerous nuclear pores.
(b) Electron micrograph of the nucleus of the alga Chlamydomonas. The dark body in the center of the nucleus is the nucleolus, and the granular material that fills the rest of the nucleus is chromatin. The nuclear envelope has paired membranes with nuclear pores; two are shown by arrows.
Figure 2–13 Chromosomes are visible in the electron microscope during mitosis. Shown here is one of the 46 human chromosomes. Every chromosome is composed of two chromatids, each consisting of tightly folded chromatin fibers. Each chromatin fiber is in turn formed by the packaging of a DNA molecule wrapped about histone proteins to form a series of nucleosomes.  (Adapted from Becker, W.M. & Deamer, D.W. (1991) The World of the Cell, 2nd edn, Fig. 13–20, The Benjamin/Cummings Publishing Company, Menlo Park, CA.)
Figure 2–14 Mitosis and cell division in animal cells. In the interphase (nondividing) nucleus (a), the chromosomes are in the form of dispersed chromatin. As mitosis begins (b), chromatin condenses into chromosomes and the mitotic spindle begins to form; centrosomes, which typically contain centriole pairs, dictate the orientation of the spindle. The nuclear envelope disintegrates and the nucleolus disappears (c), and the chromosomes align at the center of the cell (d). The chromatids of each chromosome move to opposite poles of the cell, pulled by spindle fibers attached to their centromeres (e), and a nuclear envelope forms around each new set of chromosomes (f). Finally, two daughter cells form by cell division (cytokinesis) (g). Although the same basic process occurs in all eukaryotes, there are differences in details of mitosis in plants, fungi, and protists.
The Nucleus of Eukaryotes Contains the Genome

The eukaryotic nucleus is very complex in both its structure and its biological activity, compared with the relatively simple nucleoid of prokaryotes. The nucleus contains nearly all of the cell’s DNA, typically 1,000 times more than is present in a bacterial cell; a small amount of DNA is also present in mitochondria and chloroplasts. The nucleus is surrounded by a nuclear envelope, composed of two membranes separated by a narrow space and continuous with the rough endoplasmic reticulum (Fig. 2–12; see also Fig. 2–10). At intervals the two nuclear membranes are pinched together around openings (nuclear pores), which have a diameter of about 90 nm. Associated with the pores are protein structures (nuclear pore complexes), specific macromolecule transporters that allow only certain molecules to pass between the cytoplasm and the aqueous phase of the nucleus (the nucleoplasm), such as enzymes synthesized in the cytoplasm and required in the nucleoplasm for DNA replication, transcription, or repair. Messenger RNA precursors and associated proteins also pass out of the nucleus through the nuclear pore complexes, to be translated on ribosomes in the cytoplasm; the nucleoplasm contains no ribosomes.

Inside the nucleus is the nucleolus, which appears dense in electron micrographs (Fig. 2–12b) because of its high content of RNA. The nucleolus is a specific region of the nucleus, in which the DNA contains many copies of the genes encoding ribosomal RNA. To produce the large number of ribosomes needed by the cell, these genes are continually copied into RNA (transcribed). The nucleolus is the visible evidence of the transcriptional machinery and the RNA product. Ribosomal RNA produced in the nucleolus passes into the cytoplasm through the nuclear pores. The rest of the nucleus contains chromatin, so called because early microscopists found that it stained brightly with certain dyes. Chromatin consists of DNA and proteins bound tightly to the DNA, and represents the chromosomes, which are decondensed in the interphase (nondividing) nucleus and not individually visible.

Before division of the cell (cytokinesis), nuclear division (mitosis) occurs. The chromatin condenses into discrete bodies, the chromosomes (Fig. 2–13). Cells of each species have a characteristic number of chromosomes with specific sizes and shapes. The protist Tetrahymena has 4; cabbage has 20, humans have 46, and the plant Ophioglossum, about 1,250! Usually each cell has two copies of each chromosome; such cells are called diploid. Gametes (egg and sperm, for example) produced by meiosis (Chapter 24) have only one copy of each chromosome and are called haploid. During sexual reproduction, two haploid gametes combine to regenerate a diploid cell in which each chromosome pair consists of a maternal and a paternal chromosome.

Chromosomes and chromatin are composed of DNA and a family of positively charged proteins, histones, which associate strongly with DNA by ionic interactions with its many negatively charged phosphate groups. About half of the mass of chromatin is DNA and half is histones. When DNA replicates prior to cell division, large quantities of histones are also synthesized to maintain this 1:1 ratio. The histones and DNA associate in complexes called nucleosomes, in which the DNA strand winds around a core of histone molecules (Fig. 2–13). The DNA of a single human chromosome forms about a million nucleosomes; nucleosomes associate to form very regalar and compact supramolecular complexes. The resulting chromatin fibers, about 30 nm in diameter, condense further by forming a series of looped regions, which cluster with adjacent looped regions to form the chromosomes visible during cell division. This tight packing of DNA into nucleosomes achieves a remarkable condensation of the DNA molecules. The DNA in the chromosomes of a single diploid human cell would have a combined length of about 2 m if fully stretched as a DNA double helix, but the combined length of all 46 chromosomes is only about 200 nm.

Before the beginning of mitosis, each chromosome is duplicated to form paired, identical chromatids, each of which is a double helix of DNA. During mitosis (Fig. 2–14), the two chromatids move to opposite ends (poles) of the cell, each becoming a new chromosome. Small cylindrical particles called centrioles, composed of the protein tubulin, provide the spatial organization for the migration of chromatids to opposite ends of the dividing cell. To allow the separation of chromatids, the nuclear envelope breaks down, dispersing into membrane vesicles. When the separation of the two sets of chromosomes is complete, a nuclear envelope derived from the endoplasmic reticulum re-forms around each set. Finally, the two halves of the cell are separated by cytokinesis, and each daughter cell has a complete diploid complement of chromosomes. After mitosis is complete the chromosomes decondense to form dispersed chromatin, and the nucleoli, which disappeared early in mitosis, reappear.
Figure 2–15 Structure of a mitochondrion. This electron micrograph of a mitochondrion shows the smooth outer membrane and the numerous infoldings of the inner membrane, called cristae. (Note the extensive rough endoplasmic reticulum surrounding the mitochondrion.)
Mitochondria Are the Power Plants of Aerobic Eukaryotic Cells

Mitochondria (singular, mitochondrion) are very conspicuous in the cytoplasm of most eukaryotic cells (Fig. 2–15). These membrane-bounded organelles vary in size, but typically have a diameter of about 1 μm, similar to that of bacterial cells. Mitochondria also vary widely in shape, number, and location, depending on the cell type or tissue function. Most plant and animal cells contain several hundred to a thousand mitochondria. Generally, cells in more metabolically active tissues devote a larger proportion of their volume to mitochondria.

Each mitochondrion has two membranes. The outer membrane is unwrinkled and completely surrounds the organelle. The inner membrane has infoldings called cristae, which give it a large surface area. The inner compartment of mitochondria, the matrix, is a very concentrated aqueous solution of many enzymes and chemical intermediates involved in energy-yielding metabolism. Mitochondria contain many enzymes that together catalyze the oxidation of organic nutrients by molecular oxygen (O2); some of these enzymes are in the matrix and some are embedded in the inner membrane. The chemical energy released in mitochondrial oxidations is used to generate ATP, the major energy-carrying molecule of cells. In aerobic cells, mitochondria are the

principal producers of ATP, which diffuses to all parts of the cell and provides the energy for cellular work.

Unlike other membranous structures such as lysosomes, Golgi complexes, and the nuclear envelope, mitochondria are produced only by division of previously existing mitochondria; each mitochondrion contains its own DNA, RNA, and ribosomes. Mitochondrial DNA codes for certain proteins specific to the mitochondrial inner membrane, but other mitochondrial proteins are encoded in nuclear DNA. This and other evidence supports the theory that mitochondria are the descendants of aerobic bacteria that lived symbiotically with early eukaryotic cells.

Figure 2–16  A chloroplast in a photosynthetic cell. The thylakoids are flattened membranous sacs that contain chlorophyll, the light-harvesting pigment.
Chloroplasts Convert Solar Energy into Chemical Energy

Plastids are specialized organelles in the cytoplasm of plants; they have two surrounding membranes. Most conspicuous of the plastids and characteristically present in all green plant cells and eukaryotic algae are the chloroplasts (Fig. 2–16). Like mitochondria, the chloroplasts may be considered power plants, with the important difference that chloroplasts use solar energy, whereas mitochondria use the chemical energy of oxidizable molecules. Pigment molecules in chloroplasts absorb the energy of light and use it to make ATP and, ultimately, to reduce carbon dioxide to form carbohydrates such as starch and sucrose. The photosynthetic process in eukaryotes and in cyanobacteria produces O2 as a byproduct of the light-capturing reactions. Photosynthetic plant cells contain both chloroplasts and mitochondria. Chloroplasts transduce energy only in the light, but mitochondria function independently of light, oxidizing carbohydrates generated by photosynthesis during daylight hours.

Chloroplasts are generally larger (diameter 5 μm) than mitochondria and occur in many different shapes. Because chloroplasts contain a high concentration of the pigment chlorophyll, photosynthetic cells are usually green, but their color depends on the relative amounts of other pigments present. These pigment molecules, which together can absorb light energy over much of the visible spectrum, are localized in the internal membranes of the chloroplast, which form stacks of closed cisternae known as thylakoids (Fig. 2–16). Like mitochondria, chloroplasts contain DNA, RNA, and ribosomes. Chloroplasts appear to have had their evolutionary origin in symbiotic ancestors of the cyanobacteria.
Figure 2–17 A plausible theory for the evolutionary origin of mitochondria and chloroplasts. It is based on a number of striking biochemical and genetic similarities between certain aerobic bacteria and mitochondria, and between certain cyanobacteria and chloroplasts. During the evolution of eukaryotic cells, the invading bacteria became symbiotic with the host cell. Ultimately the cytoplasmic bacteria became the mitochondria and chloroplasts of modern cells.
Mitochondria and Chloroplasts Probably Evolved from Endosymbiotic Bacteria

Several independent lines of evidence suggest that the mitochondria and chloroplasts of modern eukaryotes were derived during evolution from aerobic bacteria and cyanobacteria that took up endosymbiotic residence in early eukaryotic cells (Fig. 2–17; see also Fig. 2–7). Mitochondria are always derived from preexisting mitochondria, and chloroplasts from chloroplasts, by simple fission, just as bacteria multiply by fission. Mitochondria and chloroplasts are in fact semiautonomous; they contain DNA, ribosomes, and the enzymatic machinery to synthesize proteins encoded in their DNA. Sequences in mitochondrial DNA are strikingly similar to sequences in certain aerobic bacteria, and chloroplast DNA shows strong sequence homology with the DNA of certain cyanobacteria. The ribosomes found in mitochondria and chloroplasts are more similar in size, overall structure, and ribosomal RNA sequences to those of bacteria than to those in the cytoplasm of the eukaryotic cell. The enzymes that catalyze protein synthesis in these organelles also resemble those of the bacteria more closely.

If mitochondria and chloroplasts are the descendants of early bacterial endosymbionts, some of the genes present in the original freeliving bacteria must have been transferred into the nuclear DNA of the host eukaryote over the course of evolution. Neither mitochondria nor chloroplasts contain all of the genes necessary to specify all of their proteins. Most of the proteins of both organelles are encoded in nuclear genes, translated on cytoplasmic ribosomes, and subsequently imported into the organelles.
Figure 2–18 The three types of cytoplasmic filaments. The upper panels show epithelial cells photographed after treatment with antibodies that bind to and specifically stain (a) actin filaments bundled together to form "stress fibers", (b) microtubules radiating from the cell center, and (c) intermediate filaments, extending throughout the cytoplasm. For these experiments, antibodies that specifically recognize actin, tubulin, or intermediate filament proteins are covalently attached to a fluorescent compound. When the cell is viewed with a fluorescence microscope, only the stained structures are visible. The lower panels show each type of filament as visualized by electron microscopy.
The Cytoskeleton Stabilizes Cell Shape, Organizes the Cytoplasm, and Produces Motion

Several types of protein filaments visible with the electron microscope crisscross the eukaryotic cell, forming an interlocking three-dimensional meshwork throughout the cytoplasm, the cytoskeleton. There are three general types of cytoplasmic filaments: actin filaments, microtubules, and intermediate filaments (Fig. 2–18). They differ in width (from about 6 to 22 nm), composition, and specific function, but all apparently provide structure and organization to the cytoplasm and shape to the cell. Actin filaments and microtubules also help to produce the motion of organelles or of the whole cell.

Each of the cytoskeletal components is composed of simple protein subunits that polymerize to form filaments of uniform thickness. These filaments are not permanent structures; they undergo constant disassembly into their monomeric subunits and reassembly into filaments. Their locations in cells are not rigidly fixed, but may change dramatically with mitosis, cytokinesis, or changes in cell shape. All types of filaments associate with other proteins that cross-link filaments to themselves or to other filaments, influence assembly or disassembly, or move cytoplasmic organelles along the filaments.

Figure 2–19  Individual subunits of actin polymerize to form actin filaments. The protein filamin holds two filaments together where they cross at right angles. The filaments are cross-linked by another protein, fodrin, to form side-by-side aggregates or bundles.
Actin Filaments Are Ubiquitous in Eukaryotic Cells

Actin is a protein present in virtually all eukaryotes, from the protists to the vertebrates. In the presence of ATP, the monomeric protein spontaneously associates into linear, helical polymers, 6 to 7 nm in diameter, called actin filaments or microfilaments (Fig. 2–19).

The importance of actin polymerization and depolymerization is clear from the effects of cytochalasins, compounds that bind to actin and block polymerization. Cells treated with a cytochalasin lose actin filaments and their ability to carry out cytokinesis, phagocytosis, and amoeboid movement. However, chromatid separation at mitosis is not affected, ruling out an essential role for actin in this process. Compounds such as cytochalasins, which are naturally occurring poisons or specific toxins, are often very helpful in experimental studies in pinpointing the important participants in a biological process.

Cells contain proteins that bind to actin monomers or filaments and influence the state of actin aggregation (Fig. 2–19). Filamin and fodrin cross-link actin filaments to each other, stabilizing the meshwork and greatly increasing the viscosity of the medium in which the filaments are suspended; a concentrated solution of actin in the presence of filamin is a gel too viscous to pour. Large numbers of actin filaments bound to specific plasma membrane proteins lie just beneath and more or less parallel to the plasma membrane, conferring shape and rigidity on the cell surface.

Figure 2–20 Myosin molecules move along actin filaments using energy from ATP. Cytoplasmic streaming is produced in the giant green alga Nitella as myosin pulls organelles around a track of actin filaments. The chloroplasts of Nitella are located in the layer of stationary cytoplasm that lies between the actin filaments and the cell membrane.
Myosins Move along Actin Filaments Using the Energy of ATP

Actin filaments bind to a family of proteins called myosins, enzymes that use the energy of ATP breakdown to move themselves along the actin filament in one direction. The simplest members of this family, such as myosin I, have a globular head and a short tail (Fig. 2–20). The

head binds to and moves along an actin filament, driven by the breakdown of ATP. The tail region binds to the membrane of a cytoplasmic organelle, dragging the organelle behind as the myosin head moves along the actin filament. It appears likely that myosins of this type bind to various organelles, providing specific transport systems to move each type of organelle through the cytoplasm. This motion is readily seen in living cells such as the giant green alga Nitella; endoplasmic reticulum, as well as mitochondria, nucleus, and other membrane-bound organelles and vesicles, move uniformly around the cell at 50 to 75 μm/s in a process called cytoplasmic streaming (Fig. 2–20). This motion has the effect of mixing the cytoplasmic contents of the enormous algal cell much more efficiently than would occur by diffusion alone.

A larger form of myosin is found in muscle cells, and also in the cytoplasm of many nonmuscle cells. This type of myosin also has a globular head that binds to and moves along actin filaments in an ATP-driven reaction, but it has a longer tail, which permits myosin molecules to associate side by side to form thick filaments (see Fig. 7–31). Contractile systems composed of actin and myosin occur in a wide variety of organisms, from slime molds to humans. Actin–myosin complexes form the contractile ring that squeezes the cytoplasm in two during cytokinesis in all eukaryotes. In multicellular animals, muscle cells are filled with highly organized arrays of actin (thin) filaments and myosin (thick) filaments, which produce a coordinated contractile force by ATP-driven sliding of actin filaments past stationary myosin filaments.

Figure 2–21  Microtubules are formed from dimers of the proteins α- and β-tubulin. Colchicine blocks the assembly of microtubules, and can be used to arrest mitosis in cells.
Figure 2–22 Kinesin and dynein are ATP-driven molecular engines that move along microtubular "rails".
Microtubules Are Rigid, Hollow Rods Composed of Tubulin Subunits

Like actin filaments, microtubules form spontaneously from their monomeric subunits, but the polymeric structure of microtubules is slightly more complex. Dimers of α- and β-tubulin form linear polymers (protofilaments), 13 of which associate side by side to form the hollow microtubule, about 22 nm in diameter (Fig. 2–21). Most microtubules undergo continual polymerization and depolymerization in cells by addition of tubulin subunits primarily at one end and dissociation at the other. Microtubules are present throughout the cytoplasm, but are concentrated in specific regions at certain times. For example, when sister chromatids move to opposite poles of a dividing cell during mitosis, a highly organized array of microtubules (the mitotic spindle; Fig. 2–14) provides the framework and probably the motive force for the separation of chromatids. Colchicine, a poisonous alkaloid from meadow saffron, prevents tubulin polymerization. Colchicine treatment reversibly blocks the movement of chromatids during mitosis, demonstrating that microtubules are required for this process.

Microtubules, like actin filaments, associate with a variety of proteins that move along them, form cross-bridges, or influence their state of polymerization. Kinesin and cytoplasmic dynein, proteins found in the cytoplasm of many cells, bind to microtubules and move along them using the energy of ATP to drive their motion (Fig. 2–22). Each protein is capable of associating with specific organelles and pulling them along the microtubule over long distances at rates of about 1μm/s. The beating motion of cilia and eukaryotic flagella also involves dynein and microtubules.

Figure 2–23 Cilia and eukaryotic flagella have the same architecture: nine microtubule doublets surround a central pair of microtubules. Cross section of cilia shows the 9 + 2 arrangement of microtubules.
The Motion of Cilia and Flagella Results from Movement of Dynein along Microtubules

Cilia and flagella, motile structures extending from the surface of many protists and certain cells of animals and plants, are all constructed on the same microtubule-based architectural plan (Fig. 2–23). (Although they bear the same name, the flagella of bacteria (p. 28) are completely different in structure and in action from the flagella of eukaryotes.) Eukaryotic cilia and flagella, which are sheathed in an extension of the plasma membrane, contain nine fused pairs of microtubules arranged around two central microtubules (the 9 + 2 arrangement; Fig. 2–23). Ciliary and flagellar motion results from the coordinated sliding of outer doublet microtubules relative to their neighbors, driven by ATP. The motions of cilia and flagella propel protists through their surrounding medium, in search of food, or light, or some condition essential to their survival. Sperm are also propelled by flagellar beating. Ciliated cells in tissues such as the trachea and oviduct move extracellular fluids past the surface of the ciliated tissue.

The contraction of skeletal muscle, the propelling action of cilia and flagella, and the intracellular transport of organelles all rely on the same fundamental mechanism: the splitting of ATP by proteins such as kinesin, myosin, and dynein drives sliding motion along microfilaments or microtubules.

Intermediate Filaments Provide Structure in the Cytoplasm

The third type of cytoplasmic filament is a family of structures with dimensions (diameter 8 to 10 nm) intermediate between actin filaments and microtubules. Several different types of monomeric protein subunits form intermediate filaments. Some cells contain large amounts of one type; some types of intermediate filament are absent from certain cells; and some cell types apparently lack intermediate filaments altogether. As is the case for actin filaments and microtubules, intermediate filament formation is reversible, and the cytoplasmic distribution of these structures is subject to regulated changes.

The function of intermediate filaments is probably to provide internal mechanical support for the cell and to position its organelles. Vimentin (Mr 57,000) is the monomeric subunit of the intermediate filaments found in the endothelial cells that line blood vessels, and in adipocytes (fat cells). Vimentin fibers appear to anchor the nucleus and fat droplets in specific cellular locations. Intermediate filaments composed of desmin (Mr 55,000) hold the Z disks of striated muscle tissue in place. Neurofilaments are constructed of three different protein subunits (Mr 70,000, 150,000, and 210,000), and provide rigidity to the long extensions (axons) of neurons. In the glial cells that surround neurons, intermediate filaments are constructed from glial fibrillary acidic protein (Mr 50,000).

The intermediate filaments composed of keratins, a family of structural proteins, are particularly prominent in certain epidermal cells of vertebrates, and form covalently cross-linked meshworks that persist even after the cell dies. Hair, fingernails, and feathers are among the structures composed primarily of keratins.

The Cytoplasm Is Crowded, Highly Ordered, and Dynamic

The picture that emerges from this brief survey is of a eukaryotic cell with a cytoplasm crisscrossed by a meshwork of structural fibers, throughout which extends a complex system of membrane-bounded compartments (see Fig. 2–8). Both the filaments and the organelles are dynamic: the filaments disassemble and reassemble elsewhere; membranous vesicles bud from one organelle, move to and join another. Transport vesicles, mitochondria, chloroplasts, and other organelles move through the cytoplasm along protein filaments, drawn by kinesin, cytoplasmic dynein, myosin, and perhaps other similar proteins. Exocytosis and endocytosis provide paths between the cell interior and the surrounding medium, allowing for the secretion of proteins and other components produced within the cell and the uptake of extracellular components. The intracellular membrane systems segregate specific metabolic processes, and provide surfaces on which certain enzyme-catalyzed reactions occur.

Although complex, this organization of the cytoplasm is far from random. The motion and positioning of organelles and cytoskeletal elements are under tight regulation, and at certain stages in a eukaryotic cell’s life, dramatic, finely orchestrated reorganizations occur, such as spindle formation, chromatid migration to the poles, and nuclear envelope disintegration and re-formation during mitosis. The interactions between the cytoskeleton and organelles are noncovalent, reversible, and subject to regulation in response to various intracellular and extracellular signals. Cytoskeletal rearrangements are modulated by Ca2+ and by a variety of proteins.

Figure 2–24 A tissue such as liver is mechanically homogenized to break cells and disperse their contents in an aqueous buffer. The large and small particles in this suspension can be separated by centrifugation at different speeds (a), or particles of different density can be separated by isopycnic centrifugation (b).
In isopycnic centrifugation, a centrifuge tube is filled with a solution, the density of which increases from top to bottom; some solute such as sucrose is dissolved at different concentrations to produce this density gradient. When a mixture of organelles is layered on top of the density gradient and the tube is centrifuged at high speed, individual organelles sediment until their buoyant density exactly matches that in the gradient. Each layer can be collected separately.
Organelles Can Be Isolated by Centrifugation

A major advance in the biochemical study of cells was the development of methods for separating organelles from the cytosol and from each other. In a typical cellular fractionation, cells or tissues are disrupted by gentle homogenization in a medium containing sucrose (about 0.2 M). This treatment ruptures the plasma membrane but leaves most of the organelles intact. (The sucrose creates a medium with an osmotic pressure similar to that within organelles; this prevents diffusion of water into the organelles, which would cause them to swell, burst, and spill their contents.)

Organelles such as nuclei, mitochondria, and lysosomes differ in size and therefore sediment at different rates during centrifugation. They also differ in specific gravity, and they "float" at different levels in a density gradient (Fig. 2–24). Differential centrifugation results in a rough fractionation of the cytoplasmic contents, which may be further purified by isopycnic centrifugation. In this procedure, organelles of different buoyant densities (the result of different ratios of lipid and protein in each type of organelle) are separated on a density gradient. By carefully removing material from each region of the gradient and observing it with a microscope, the biochemist can establish the position of each organelle and obtain purified organelles for further study. In this way it was established, for example, that lysosomes contain degradative enzymes, mitochondria contain oxidative enzymes, and chloroplasts contain photosynthetic pigments. The isolation of an organelle enriched in a certain enzyme is often the first step in the purification of that enzyme.
In Vitro Studies May Overlook Important Interactions among Molecules

One of the most effective approaches to understanding a biological process is to study purified individual molecules such as enzymes, nucleic acids, or structural proteins. The purified components are amenable to detailed characterization in vitro; their physical properties and catalytic activities can be studied without "interference" from other molecules present in the intact cell. Although this approach has been remarkably revealing, it must always be remembered that the inside of a

cell is quite different from the inside of a test tube. The "interfering" components eliminated by purification may be critical to the biological function or regulation of the molecule purified. In vitro studies of pure enzymes are commonly done at very low enzyme concentrations in thoroughly stirred aqueous solutions. In the cell, an enzyme is dissolved or suspended in a gel-like cytosol with thousands of other proteins, some of which bind to that enzyme and influence its activity. Within cells, some enzymes are parts of multienzyme complexes in which reactants are channeled from one enzyme to another without ever entering the bulk solvent. Diffusion is hindered in the gel-like cytosol, and the cytosolic composition varies in different regions of the cell. In short, a given molecule may function somewhat differently within the cell than it does in vitro. One of the central challenges of biochemistry is to understand the influences of cellular organization and macromolecular associations on the function of individual enzymes – to understand function in vivo as well as in vitro.
Figure 2–25 A gallery of differentiated cells. (a) Secretory cells of the pancreas, with an extensive endoplasmic reticulum. (b) Portion of a skeletal muscle cell, with organized actin and myosin filaments.
(c) Collenchyma cells of a plant stem. (d) Rabbit sperm cells, with long flagella for motility. (e) Human erythrocyte. (f) Human embryo at the two-celled stage.

Figure 2–26 Three types of junctions between cells.
(a) Tight junctions produce a seal between adjacent cells. (b) Desmosomes, typical of plant cells, weld adjacent cells together and are reinforced by various cytoskeletal elements. (c) Gap junctions allow ions and electric currents to flow between adjacent cells.
Evolution of Multicellular Organisms and Cellular Differentiation

All modern unicellular eukaryotes – the protists – contain the organelles and mechanisms that we have described, indicating that these organelles and mechanisms must have evolved relatively early. The protists are extraordinarily versatile. The ciliated protist Paramecium, for example, moves rapidly through its aqueous surroundings by beating its cilia; senses mechanical, chemical, and thermal stimuli from its environment, and responds by changing its path; finds, engulfs, and digests a variety of food organisms, and excretes the indigestible fragments; eliminates excess water that leaks through its membrane; and finds and mates with sexual partners. Nonetheless, being unicellular has its disadvantages. Paramecia probably live out their lives in a very small region of the pond in which they began life, because their motility is limited by the small thrust of their microscopic cilia, and their ability to detect a better environment at a distance is limited by the short range of their sensory apparatus.

At some later stage of evolution, unicellular organisms found it advantageous to cluster together, thereby acquiring greater motility, efficiency, or reproductive success than their free-living single-celled competitors. Further evolution of such clustered organisms led to permanent associations among individual cells and eventually to specialization within the colony – to cellular differentiation.

The advantages of cellular specialization led to the evolution of ever more complex and highly differentiated organisms, in which some cells carried out the sensory functions, others the digestive, photosynthetic, or reproductive functions. Many modern multicellular organisms contain hundreds of different cell types, each specialized for some function that supports the entire organism. Fundamental mechanisms that evolved early have been further refined and embellished through evolution. The simple mechanism responsible for the motion of myosin along actin filaments in slime molds has been conserved and elaborated in vertebrate muscle cells, which are literally filled with actin, myosin, and associated proteins that regulate muscle contraction. The same basic structure and mechanism that underlie the beating motion of cilia in Paramecium and flagella in Chlamydomonas are employed by the highly differentiated vertebrate sperm cell. Figure 2–25 illustrates the range of cellular specializations encountered in multicellular organisms.

The individual cells of a multicellular organism remain delimited by their plasma membranes, but they have developed specialized surface structures for attachment to and communication with each other (Fig. 2–26). At tight junctions, the plasma membranes of adjacent cells are closely apposed, with no extracellular fluid separating them. Desmosomes (occurring only in plant cells) hold two cells together; the small extracellular space between them is filled with fibrous, presumably adhesive, material. Gap junctions provide small, reinforced openings between adjacent cells, through which electric currents, ions, and small molecules can pass. In higher plants, plasmodesmata form channels resembling gap junctions; they provide a path through the cell wall for the movement of small molecules between adjacent cells. Each of these junctions is reinforced by membrane proteins or cytoskeletal filaments. The type of junction(s) between neighboring cells varies from tissue to tissue.

Figure 2–27 Infection of a bacterial cell by a bacteriophage (left), and of an animal cell by a virus (right) results in the formation of many copies of the infecting virus.
Figure 2–28 The structures of several viruses, viewed with the electron microscope. Turnip yellow mosaic virus (small, spherical particles), tobacco mosaic virus (long cylinders), and bacteriophage T4 (shaped like a hand mirror).
Figure 2–29 Human immunodeficiency viruses (HIV), the causative agent of AIDS, leaving an infected T lymphocyte of the immune system.
Viruses: Parasites of Cells

Viruses are supramolecular complexes that can replicate themselves in appropriate host cells. They consist of a nucleic acid (DNA or RNA) molecule surrounded by a protective shell, or capsid, made up of protein molecules and, in some cases, a membranous envelope. Viruses exist in two states. Outside the host cells that formed them, viruses are simply nonliving particles called virions, which are regular in size, shape, and composition and can be crystallized. Once a virus or its nucleic acid component gains entry into a specific host cell, it becomes an intracellular parasite. The viral nucleic acid carries the genetic message specifying the structure of the intact virion. It diverts the host cell’s enzymes and ribosomes from their normal cellular roles to the manufacture of many new daughter viral particles. As a result, hundreds of progeny viruses may arise from the single virion that infected the host cell (Fig. 2–27). In some host–virus systems, the progeny virions escape through the host cell’s plasma membrane. Other viruses cause cell lysis (membrane breakdown and host cell death) as they are released.

A different type of response results from some viral infections, in which viral DNA becomes integrated into the host’s chromosome and is replicated with the host’s own genes. Integrated viral genes may have little or no effect on the host’s survival, but they often cause profound changes in the host cell’s appearance and activity.

Hundreds of different viruses are known, each more or less specific for a host cell (Table 2–3), which may be an animal, plant, or bacterial cell. Viruses specific for bacteria are known as bacteriophages, or simply phages (Greek phagein, "to eat"). Some viruses contain only one kind of protein in their capsid – the tobacco mosaic virus, for example, a simple plant virus and the first to be crystallized. Other viruses contain dozens or hundreds of different kinds of proteins. Even some of these large and complex viruses have been crystallized, and their detailed molecular structures are known (Fig. 2–28). Viruses differ greatly in size. Bacteriophage ΦX174, one of the smallest, has a diameter of 18 nm. Vaccinia virus is one of the largest; its virions are almost as large as the smallest bacteria. Viruses also differ in shape and complexity of structure. The human immunodeficiency virus (HIV) (Fig. 2–29) is relatively simple in structure, but devastating in action; it causes AIDS.

Table 2–3 summarizes the type and size of the nucleic acid components of a number of viruses. Some viruses are highly pathogenic in humans; for example, those causing poliomyelitis, influenza, herpes, hepatitis, AIDS, the common cold, infectious mononucleosis, shingles, and certain types of cancer.

Biochemistry has profited enormously from the study of viruses, which has provided new information about the structure of the genome, the enzymatic mechanisms of nucleic acid synthesis, and the regulation of the flow of genetic information.

Cells, the structural and functional units of living organisms, are of microscopic dimensions. Their small size, combined with convolutions of their surfaces, results in high surface-to-volume ratios, facilitating the diffusion of fuels, nutrients, and waste products between the cell and its surroundings. All cells share certain features: DNA containing the genetic information, ribosomes, and a plasma membrane that surrounds the cytoplasm. In eukaryotes the genetic material is surrounded by a nuclear envelope; prokaryotes have no such membrane.

The plasma membrane is a tough, flexible permeability barrier, which contains numerous transporters as well as receptors for a variety of extracellular signals. The cytoplasm consists of the cytosol and organelles. The cytosol is a concentrated solution of proteins, RNA, metabolic intermediates and cofactors, and inorganic ions, in which are suspended various particles. Ribosomes are supramolecular complexes on which protein synthesis occurs; bacterial ribosomes are slightly smaller than those of eukaryotic cells, but are similar in structure and function.

Certain organisms, tissues, and cells offer advantages for biochemical studies. E. coli and yeast can be cultured in large quantities, have short generation times, and are especially amenable to genetic manipulation. The specialized functions of liver, muscle, and fat tissue, and of erythrocytes, make them attractive for the study of specific processes.

The first living cells were prokaryotic and anaerobic; they probably arose about 3.5 billion years ago, when the atmosphere was devoid of oxygen. With the passage of time, biological evolution led to cells capable of photosynthesis, with O2 as a byproduct. As O2 accumulated, prokaryotic cells capable of the aerobic oxidation of fuels evolved. The two major groups of bacteria, eubacteria and archaebacteria, diverged early in evolution. The cell envelope of some types of bacteria includes layers outside the plasma membrane that provide rigidity or protection. Some bacteria have flagella for propulsion. The cytoplasm of bacteria contains no membrane-bounded organelles but does contain ribosomes and granules of nutrients, as well as a nucleoid which contains the cell’s DNA. Some photosynthetic bacteria have extensive intracellular membranes that contain light-capturing pigments.

About 1.5 billion years ago, eukaryotic cells emerged. They were larger than bacteria, and their genetic material was more complex. These early cells established symbiotic relationships with prokaryotes that lived in their cytoplasm; modern mitochondria and chloroplasts are derived from these early endosymbionts. Mitochondria and chloroplasts are intracellular organelles surrounded by a double membrane. They are the principal sites of ATP synthesis in eukaryotic, aerobic cells. Chloroplasts are found only in photosynthetic organisms, but mitochondria are ubiquitous among eukaryotes.

Modern eukaryotic cells have a complex system of intracellular membranes. This endomembrane system consists of the nuclear envelope, rough and smooth endoplasmic reticulum, the Golgi complex, transport vesicles, lysosomes, and endosomes. Proteins synthesized on ribosomes bound to the rough endoplasmic reticulum pass into the endomembrane system, traveling through the Golgi complex on their way to organelles or to the cell surface, where they are secreted by exocytosis. Endocytosis brings extracellular materials into the cell, where they can be digested by degradative enzymes in the lysosomes. In plants, the central vacuole is the site of degradative processes; it also serves as a storage depot for a variety of side products of metabolism and maintains cell turgor.

The genetic material in eukaryotic cells is organized into chromosomes, highly ordered complexes of DNA and histone proteins. Before cell division (cytokinesis), each chromosome is replicated, and the duplicate chromosomes are separated by the process of mitosis.

The cytoskeleton is an intracellular meshwork of actin filaments, microtubules, and intermediate filaments of several types. The cytoskeleton confers shape on the cell, and reorganization of cytoskeletal filaments results in the shape changes accompanying amoeboid movement and cell division. Intracellular organelles move along filaments of the cytoskeleton, propelled by proteins such as dynein, kinesin, and myosin, using the energy of ATP. Dynein and tubulin are central to the motion and strncture of cilia and ilagella, and myosin and actin are responsible for the contractile motion of skeletal muscle. The organelles can be separated by differential centrifugation and by isopycnic centrifugation.

In multicellular organisms, there is a division of labor among several types of cells. Individual cells are joined to each other by tight junctions or gap junctions, and (in plants) desmosomes or plasmodesmata. Viruses are parasites of living cells, capable of subverting the cellular machinery for their own replication.

Further Reading


Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K., & Watson, J.D. (1989) Molecular Biology of the Cell, 2nd edn, Garland Publishing, Inc., New York. 
A superb textbook on cell structure and function, covering the topics considered in this chapter, and a useful reference for many of the following chapters.

Becker, W.M. & Deamer, D.W. (1991) The World of the Cell, 2nd edn, The Benjamin/Cummings Publishing Company, Redwood City, CA. 
An excellent introductory textbook of cell biology.

Curtis, H. & Barnes, N.S. (1989) Biology, 5th edn, Worth Publishers, Inc., New York. 
A beautifully written and illustrated general biology textbook.

Darnell, J., Lodish, H., & Baltimore, D. (1990) Molecular Cell Biology, 2nd edn, Scientific American Books, Inc., New York. 
Like the book by Alberts and coauthors, a superb text useful for this and later chapters.

Prescott, D.M. (1988) Cells, Jones and Bartlett Publishers, Boston, MA. 
A short, well-illustrated introductory textbook on cell structure and function, with emphasis on structure.

Evolution of Cells

Evolution of Catalytic Function. (1987) Cold Spring Harb. Symp. Quant. Biol. 52. 
A collection of excellent papers on many aspects of molecular and cellular evolution.

Knoll, A.H. (1991) End of the proterozoic eon. Sci. Am. 265 (October), 64–73. 
Discussion of the evidence that an increase in atmospheric oxygen led to the development of multi-cellular organisms, including large animals.

Margulis, L. (1992) Symbiosis in Cell Euolution. Microbial Evolution in the Archean and Proterozoic Eons, 2nd edn, W.H. Freeman and Company, New York. 
Clear discussion of the hypothesis that mitochondria and chloroplasts are descendants of bacteria that became symbiotic with primitive eukaryotic cells.

Schopf, J.W. (1978) The evolution of the earliest cells. Sci. Am. 239 (September), 110–139. 

Vidal, G. (1984) The oldest eukaryotic cell. Sci. Am. 250 (February), 48–57. 

Structure of Cells and Organelles

Bloom, W. & Fawcett, D.W. (1986) A Textbook of Histology, 11th edn, W.B. Saunders Company, Philadelphia, PA. 
A standard textbook, containing detailed descriptions of the structures of animal cells, tissues, and organs.

de Duve, C. (1984) A Guided Tour of the Living Cell, Scientific American Books, Inc., New York. 
An easy-to-read, well-illustrated description of the structure and functions of the organelles of the eukaryotic cell.

Margulis, L. & Schwartz, K.V. (1987) Five Kingdoms: An Illustrated Guide to the Phyla of Life on Earth, 2nd edn, W.H. Freeman and Company, New York. 
Description of unicellular and multicellular organisms, beautifully illustrated with electron micrographs and drawings showing the diversity of structure and function.

Rothman, J.E. (1985) The compartmental organization of the Golgi apparatus. Sci. Am. 253 (September), 74–89. 


Gelfand, V. & Bershadsky, A.D. (1991) Microtubule dynamics: mechanism, regulation, and function. Annu. Rev. Cell Biol. 7, (September), 93–116. 

Organization of the Cytoplasm. (1981) Cold Spring Harb. Symp. Quant. Biol. 46. 
More than 90 excellent papers on microtubules, microfilaments, and intermediate filaments and their biological roles.

Schroer, T.A. & Sheetz, M.P. (1991) Functions of microtubule-based motors. Annu. Rev. Physiol. 53, 629–652. 

Steinert, P.M. & Parry, D.A.D. (1985) Intermediate filaments: conformity and diversity of expression and structure. Annu. Rev. Cell Biol. 1, 41–65. 

Stossel, T.P. (1989) From signal to pseudopod: how cells control cytoplasmic actin assembly. J. Biol. Chem. 264, 18261–18264. 

Vale, R.D. (1990) Microtubule-based motor proteins. Curr. Opinion Cell Biol. 2, 15–22. 

Vallee, R.B. & Shpetner, H.S. (1990) Motor proteins of cytoplasmic microtubules. Annu. Rev. Biochem. 59, 909–932. 


Some problems on the contents of Chapter 2 follow. They involve simple geometrical and numerical relationships concerning cell structure and activities. (For your reference in solving these problems, please see the tables printed on the inside of the back cover.) Each problem has a title for easy reference and discussion.

1. The Size of Cells and Their Components  Given their approximate diameters, calculate the approximate number of (a) hepatocytes (diameter 20 μm), (b) mitochondria (1.5 μm), and (c) actin molecules (3.6 nm) that can be placed in a single layer on the head of a pin (diameter 0.5 mm). Assume each structure is spherical. The area of a circle is πr2, where π = 3.14.

2. Number of Solute Molecules in the Smallest Known Cells  Mycoplasmas are the smallest known cells. They are spherical and have a diameter of about 0.33 μm. Because of their small size they readily pass through filters designed to trap larger bacteria. One species, Mycoplasma pneumoniae, is the causative organism of the disease primary atypical pneumonia.

(a) D-Glucose is the major energy-yielding nutrient of mycoplasma cells. Its concentration within such cells is about 1 mM. Calculate the number of glucose molecules in a single mycoplasma cell. Avogadro’s number, the number of molecules in 1 mol of a nonionized substance, is 6.02 × 1023. The volume of a sphere is 4πr3/3.

(b) The first enzyme required for the energy-yielding metabolism of glucose is hexokinase (Mr 100,000). Given that the intracellular fluid of mycoplasma cells contains 10 g of hexokinase per liter, calculate the molar concentration of hexokinase.

3. Components of E. coli  E. coli cells are rodshaped, about 2 μm long and 0.8 μm in diameter. The volume of a cylinder is πr2h, where h is the height of the cylinder.

(a) If the average density of E. coli (mostly water) is 1.1 × 103 g/L, what is the weight of a single cell?

(b) The protective cell wall of E. coli is 10 nm thick. What percentage of the total volume of the bacterium does the wall occupy?

(c) E. coli is capable of growing and multiplying rapidly because of the inclusion of some 15,000 spherical ribosomes (diameter 18 nm) in each cell, which carry out protein synthesis. What percentage of the total cell volume do the ribosomes occupy?

4. Genetic Information in E. coli DNA  The genetic information contained in DNA consists of a linear sequence of successive code words, known as codons. Each codon is a specific sequence of three nucleotides (three nucleotide pairs in doublestranded DNA), and each codon codes for a single amino acid unit in a protein. The molecular weight of an E. coli DNA molecule is about 2.5 × 109. The average molecular weight of a nucleotide pair is 660, and each nucleotide pair contributes 0.34 nm to the length of DNA.

(a) Calculate the length of an E. coli DNA molecule. Compare the length of the DNA molecule with the actual cell dimensions. How does the DNA molecule fit into the cell?

(b) Assume that the average protein in E. coli consists of a chain of 400 amino acids. What is the maximum number of proteins that can be coded by an E. coli DNA molecule?

5. The High Rate of Bacterial Metabolism  Bacterial cells have a much higher rate of metabolism than animal cells. Under ideal conditions some bacteria will double in size and divide in 20 min, whereas most animal cells require 24 h. The high rate of bacterial metabolism requires a high ratio of surface area to cell volume.

(a) Why would the surface-to-volume ratio have an effect on the maximum rate of metabolism?

(b) Calculate the surface-to-volume ratio for the spherical bacterium Neisseria gonorrhoeae (diameter 0.5 μm), responsible for the disease gonorrhea. Compare it with the surface-to-volume ratio for globular amoeba, a large eukaryotic cell of diameter 150 μm. The surface area of a sphere is 4πr2.

6. A Strategy to Increase the Surface Area of Cells  Certain cells whose function is to absorb nutrients, e.g., the cells lining the small intestine or the root hair cells of a plant, are optimally adapted to their role because their exposed surface area is increased by microvilli. Consider a spherical epithelial cell (diameter 20 μm) lining the small intestine. Since only a part of the cell surface faces the interior of the intestine, assume that a "patch" corresponding to 25% of the cell area is covered with microvilli. Furthermore, assume that the microvilli are cylinders 0.1 μm in diameter, 1.0 μm long, and spaced in a regular grid 0.2 μm on center.

(a) Calculate the number of microvilli on the patch.

(b) Calculate the surface area of the patch, assuming it has no microvilli.

(c) Calculate the surface area of the patch, assuming it does have microvilli.

(d) What percentage improvement of the absorptive capacity (reflected by the surface-tovolume ratio) does the presence of microvilli provide?

7. Fast Axonal Transport  Some neurons have long, thin extensions (axons) as long as 2 m. Small membrane vesicles carrying materials essential to axonal function move along microtubules from the cell body to the tip of the axon by kinesin-dependent "fast axonal transport." If the average velocity of a vesicle is 1 μm/s, how long does it take a vesicle to move the 2 m from cell body to axonal tip? What are the possible advantages of this ATP-dependent process over simple diffusion to move materials to the axonal tip?

8. Toxic Effects of Phalloidin  Phalloidin is a toxin produced by the mushroom Amanita phalloides. It binds specifically to actin microfilaments and blocks their disassembly. Cytochalasin B is another toxin, which blocks microfilament assembly from actin monomers (see p. 42).

(a) Predict the effect of phalloidin on cytokinesis, phagocytosis, and amoeboid movement, given the effects of cytochalasins on these processes.

(b) A specific antibody (a protein of Mr ≈ 150,000) binds actin tightly and is found to block microfilament assembly in vitro (in the test tube). Would you expect this antibody to mimic the effects of cytochalasin in vivo (in living cells)?

9. Osmotic Breakage of Organelles  In the isolation of cytosolic enzymes, cells are often broken in the presence of 0.2 M sucrose to prevent osmotic swelling and bursting of the intracellular organelles. If the desired enzymes are in the cytosol, why is it necessary to be concerned about possible damage to particulate organelles?
The chemical composition of living material, such as this jellyfish, differs from that of its physical environment, which for this organism is salt water.
Chapter 3
Biochemistry aims to explain biological form and function in chemical terms. One of the most fruitful approaches to understanding biological phenomena has been to purify an individual chemical component, such as a protein, from a living organism and to characterize its chemical structure or catalytic activity. As we begin the study of biomolecules and their interactions, some basic questions deserve attention. What chemical elements are found in cells? What kinds of molecules are present in living matter? In what proportions do they occur? How did they come to be there? In what ways are the kinds of molecules found in living cells especially suited to their roles?

We review here some of the chemical principles that govern the properties of biological molecules: the covalent bonding of carbon with itself and with other elements, the functional groups that occur in common biological molecules, the three-dimensional structure and stereochemistry of carbon compounds, and the common classes of chemical reactions that occur in living organisms. Next, we discuss the monomeric units and the contribution of entropy to the free-energy changes of reactions in which these units are polymerized to form macromolecules. Finally, we consider the origin of the monomeric units from simple compounds in the earth’s atmosphere during prebiological times – that is, chemical evolution.

Figure 3–1 Elements essential to animal life and health. Bulk elements (shaded orange) are structural components of cells and tissues and are required in the diet in gram quantities daily. For trace elements (shaded yellow), the requirements are much smaller: for humans, a few milligrams per day of Fe, Cu, and Zn, even less of the others. The elemental requirements for plants and microorganisms are very similar to those shown here.
Chemical Composition

By the beginning of the nineteenth century, it had become clear to chemists that the composition of living matter is strikingly different from that of the inanimate world. Antoine Lavoisier (1743–1794) noted the relative chemical simplicity of the "mineral world", and contrasted it with the complexity of the "plant and animal worlds"; the latter, he knew, were composed of compounds rich in the elements carbon, oxygen, nitrogen, and phosphorus. The development of organic chemistry preceded, and provided invaluable insights for, the development of biochemistry.

We will briefly review some fundamental concepts of organic chemistry: the nature of bonding between atoms of carbon and of hydrogen, oxygen, and nitrogen; the functional groups that result from these combinations; and the diversity of organic compounds that are derived from these elements.

Figure 3–2 Covalent bonding. Two atoms with unpaired electrons in their outer shells can form covalent bonds with each other by sharing electron pairs. Atoms participating in covalent bonding tend to fill their outer electron shells.
Living Matter Is Composed Mostly of the Lighter Elements

Only about 30 of the more than 90 naturally occurring chemical elements are essential to living organisms. Most of the elements in living matter have relatively low atomic numbers; only five have atomic numbers above that of selenium, 34 (Fig. 3–1). The four most abundant elements in living organisms, in terms of the percentage of the total number of atoms, are hydrogen, oxygen, nitrogen, and carbon, which together make up over 99% of the mass of most cells. They are the lightest elements capable of forming one, two, three, and four bonds, respectively (Fig. 3–2). In general, the lightest elements form the strongest bonds. Six of the eight most abundant elements in the

human body are also among the nine most abundant elements in seawater (Table 3–1), and several of the elements abundant in humans are components of the atmosphere and were probably present in the atmosphere before the appearance of life on earth. Primitive seawater was most likely the liquid medium in which living organisms first arose, and the primitive atmosphere was probably a source of methane, ammonia, water, and hydrogen, the starting materials for the evolution of life. The trace elements (Fig. 3–1) represent a miniscule fraction of the weight of the human body, but all are absolutely essential to life, usually because they are essential to the function of specific enzymes (Table 3–2).

Figure 3–3 Versatility of carbon in forming covalent single, double, and triple bonds (in red), particularly between carbon atoms. triple bonds occur only rarely in biomolecules.
Biomolecules Are Compounds of Carbon

The chemistry of living organisms is organized around the element carbon, which accounts for more than one-half the dry weight of cells. In methane (CH4), a carbon atom shares four electron pairs with four hydrogen atoms; each of the shared electron pairs forms a single bond. Carbon can also form single and double bonds to oxygen and nitrogen atoms (Fig. 3–3). Of greatest significance in biology is the ability of carbon atoms to share electron pairs with each other to form very stable carbon–carbon single bonds. Each carbon atom can form single bonds with one, two, three, or four other carbon atoms. Two carbon atoms also can share two (or three) electron pairs, thus forming carbon–carbon double (or triple) bonds (Fig. 3–3). Covalently linked carbon atoms can form linear chains, branched chains, and cyclic and cagelike structures. To these carbon skeletons are added groups of other atoms, called functional groups, which confer specific chemical properties on the molecule. Molecules containing covalently bonded carbon backbones are called organic compounds; they occur in an almost limitless variety. Most biomolecules are organic compounds; we can therefore infer that the bonding versatility of carbon was a major factor in the selection of carbon compounds for the molecular machinery of cells during the origin and evolution of living organisms.

Figure 3–4 (a) Carbon atoms have a characteristic tetrahedral arrangement of their four single bonds, which are about 0.154 nm long and at an angle of 109.5° to each other. (b) Carbon–carbon single bonds have freedom of rotation, shown for the compound ethane (CH3–CH3). (c) Carbon–carbon double bonds are shorter and do not allow free rotation. The single bonds on each doubly bonded carbon make an angle of 120° with each other. The two doubly bonded carbons and the atoms designated A, B, X, and Y all lie in the same rigid plane.
Organic Compounds Have Specific Shapes and Dimensions

The four covalent single bonds that can be formed by a carbon atom are arranged tetrahedrally, with an angle of about 109.5° between any two bonds (Fig. 3–4) and an average length of 0.154 nm. There is free rotation around each carbon–carbon single bond unless very large or highly charged groups are attached to both carbon atoms, in which case rotation may be restricted. A carbon–carbon double bond is shorter (about 0.134 nm long) and rigid and allows little rotation about its axis. (Fig. 3–4). No other chemical element can form molecules of such widely different sizes and shapes or with such a variety of functional groups.

Figure 3–5 Some functional groups frequently encountered in biomolecules. All groups are shown in their uncharged (un-ionized) form.
Figure 3–6 Representative biomolecules with multiple functional groups. Note that secondary (s) and tertiary (t) amino groups have, respectively, one and two of their amino hidrogens replaced by other groups.
Functional Groups Determine Chemical Properties

Most biomolecules can be regarded as derivatives of hydrocarbons, compounds with a covalently linked carbon backbone to which only hydrogen atoms are bonded. The backbones of hydrocarbons are very stable. The hydrogen atoms may be replaced by a variety of functional groups to yield different families of organic compounds. Typical families of organic compounds are the alcohols, which have one or more hydroxyl groups; amines, which have amino groups; aldehydes and ketones, which have carbonyl groups; and carboxylic acids, which have carboxyl groups (Fig. 3–5).

Many biomolecules are polyfunctional, containing two or more different kinds of functional groups (Fig. 3–6), each with its own chemical characteristics and reactions. Amino acids, an important family of molecules that serve primarily as monomeric subunits of proteins, contain at least two different kinds of functional groups: an amino group and a carboxyl group, as shown for histidine in Figure 3–6. The ability of an amino acid to condense (see Fig. 3–14e) with other amino acids to form proteins is dependent on the chemical properties of these two functional groups.
Three-Dimensional Structure

Although the covalent bonds and functional groups of biomolecules are central to their function, they do not tell the whole story. The arrangement in three-dimensional space of the atoms of a biomolecule is also crucially important. Compounds of carbon can often exist in two or more chemically indistinguishable three-dimensional forms, only one of which is biologically active. This specificity for one particular molecular configuration is a universal feature of biological interactions. All biochemistry is three-dimensional.

Figure 3–7 Models of the structure of the amino acid alanine. (a) Structural formula in perspective form. The symbol ◅ represents a bond in which the atom at the wide end projects out of the plane of the paper, toward the reader; dashes represent a bond extending behind the plane of the paper. (b) Ball-and-stick model, showing relative bond lengths and the bond angles. The balls indicate the approximate size of the atomic nuclei.
(c) Space-filling model, in which each atom is shown having its correct van der Waals radius (see Table 3–3).
Figure 3–8 Complementary fit of a substrate molecule to the active or catalytic site on an enzyme molecule. The enzyme shown here is chymotrypsin, an enzyme that acts in the intestine to degrade dietary protein. Its substrate (shown in red) fits into a groove at the active site of the enzyme.
Each Cellular Component Has a Characteristic Three-Dimensional Structure

Biomolecules have characteristic sizes and three-dimensional structures, which derive from their backbone structures and their substituent functional groups. Figure 3–7 shows three ways to illustrate the three-dimensional structures of molecules. The perspective diagram specifies unambiguously the three-dimensional structure (stereochemistry) of a compound. Bond angles and center-to-center bond lengths are best represented with ball-and-stick models, whereas the outer contours of molecules are better represented by space-filling models. In space-filling models, the radius of each atom is proportional to its van der Waals radius (Table 3–3), and the contours of the molecule represent the outer limits of the region from which atoms of other molecules are excluded.

The three-dimensional conformation of biomolecules is of the utmost importance in their interactions; for example, in the binding of a substrate (reactant) to the catalytic site of an enzyme (Fig. 3–8), the two molecules must fit each other closely, in a complementary fashion, for biological function. Such complementarity also is required in the binding of a hormone molecule to its receptor on a cell surface, or in the recognition of an antigen by a specific antibody.

The study of the three-dimensional structure of biomolecules with precise physical methods is an important part of modern research on cell structure and biochemical function. The most informative method is x-ray crystallography. If a compound can be crystallized, the diffraction of x rays by the crystals can be used to determine with great precision the position of every atom in the molecule relative to every other atom. The structures of most small biomolecules (those with less than about 50 atoms), and of many larger molecules such as proteins, have been deduced by this means. X-ray crystallography yields a static picture of the molecule within the confines of the crystal. However, biomolecules almost never exist within cells as crystals; rather, they are dissolved in the cytosol or associated with some other component(s) of the cell. Molecules have more freedom of intramolecular motion in solution than in a crystal. In large molecules such as proteins, the small variations allowed in the three-dimensional structures of their monomeric subunits add up to extensive flexibility. Techniques such as nuclear magnetic resonance (NMR) spectroscopy complement x-ray crystallography by providing information about the three-dimensional structure of biomolecules in solution. Knowledge of the detailed three-dimensional structure of a molecule often sheds light on the mechanisms of the reactions in which the molecule participates.

Figure 3–9 Molecular asymmetry: chiral and achiral molecules. (a) When a carbon atom has four difierent substituent groups (A, B, X, Y), they can be arranged in two ways that represent nonsuperimposable mirror images of each other (enantiomers). Such a carbon atom is asymmetric and is called a chiral atom or chiral center. (b) When there are only three dissimilar groups around the carbon atom (i.e., the same group occurs twice), only one configuration in space is possible and the molecule is symmetric, or achiral. In this case the molecule is superimposable on its mirror image: the molecule on the left can be rotated counterclockwise (when looking down its vertical bond from A to C) to create the molecule on the right.
Louis Pasteur
Figure 3–10 Pasteur separated crystals of two stereoisomers of tartaric acid and showed that solutions of the separated forms each rotated polarized light to the same extent but in opposite directions. Pasteur’s dextrorotatory and levorotatory forms were later shown to be the R,R and S,S isomers shown here. For compounds with more than one chiral center, the RS system
of nomenclature is often more useful than the D and L system described in Chapter 5. In the RS system, each group attached to a chiral carbon is assigned a priority. The priorities of some common substituents are: –OCH2 > –OH > –NH2 > –COOH > –CHO > –CH2OH > –CH3 > –H. The chiral carbon atom is viewed with the group of lowest priority pointing away from the viewer. If the priority of the other three groups decreases in counterclockwise order, the configuration is S; if in clockwise order, R. In this way each chiral carbon is designated as either R or S, and the inclusion of these designations in the name of the compound provides an unambiguous description of the stereochemistry at each chiral center.
Figure 3–11 Configurations of stereoisomers. (a) Isomers such as maleic acid and fumaric acid cannot be interconverted without breaking covalent bonds, which requires the input of much energy. (b) In the vertebrate retina, the initial event in light detection is the absorption of visible light by 11-cis-retinal. The energy of the absorbed light (about 250 kJ/mol) converts 11-cis-retinal to all-trans-retinal, triggering electrical changes in the retinal cell that lead to a nerve impulse.
Figure 3–12 Many conformations of ethane are possible because of freedom of rotation around the carbon–carbon single bond. When the front carbon atom (as viewed by the reader) and its three attached hydrogens are rotated relative to the rear carbon atom, the potential energy of the molecule rises in the fully eclipsed conformation (torsion angle 0°, 120°, etc.), then falls in the fully staggered conformation (torsion angle 60°, 180°, etc.). The energy differences are small enough to allow rapid interconversion of the two forms (millions of times per second), thus the eclipsed and staggered forms cannot be isolated separately.
Most Biomolecules Are Asymmetric

The tetrahedral arrangement of single bonds around a carbon atom confers on some organic compounds another property of great importance in biology. When four different atoms or functional groups are bonded to a carbon atom in an organic molecule, the carbon atom is said to be asymmetric; it can exist in two different isomeric forms (stereoisomers) that have different configurations in space. A special class of stereoisomers, called enantiomers, are nonsuperimposable mirror images of each other (Fig. 3–9). The two enantiomers of a compound have identical chemical properties, but differ in a characteristic physical property, the ability to rotate the plane of plane-polarized light. A solution of one enantiomer rotates the plane of such light to the right, and a solution of the other, to the left. Compounds without an asymmetric carbon atom do not rotate the plane of plane-polarized light.

Louis Pasteur, in 1843, was the first to arrive at the correct explanation for this phenomenon of optical activity. Investigating the crystalline material that accumulated in wine casks ("paratartaric acid," also called racemic acid, from Latin racemus, "grape"), he had used a fine forceps to separate two types of crystals identical in shape, but mirror images of each other (Fig. 3–10). Both proved to have all of the chemical properties of tartaric acid, but one type rotated polarized light to the left, the other, to the right, but to the same extent. He later described the experiment and its interpretation:

In isomeric bodies, the elements and the proportions in which they are combined are the same, only the arrangement of the atoms is different. . . . We know, on the one hand, that the molecular arrangements of the two tartaric acids are asymmetric, and, on the other hand, that these arrangements are absolutely identical, excepting that they exhibit asymmetry in opposite directions. Are the atoms of the dextro acid grouped in the form of a right-handed spiral, or are they placed at the apex of an irregular tetrahedron, or are they disposed according to this or that asymmetric arrangement? We do not know.*

* From Pasteur’s lecture to the Société Chimique de Paris in 1883, quoted in DuBos, R. (1976) Louis Pasteur: Free Lance of Science, p. 95, Charles Scribner’s Sons, New York.
Now we do know. X-ray crystallographic studies in 1951 confirmed that the levorotatory and dextrorotatory forms of tartaric acid are mirror images of each other, and established the absolute configuration of each (Fig. 3–10). The same approach has been used to demonstrate that the amino acid alanine exists in two enantiomeric forms (Chapter 5). The central carbon atom of the alanine molecule is bonded to four different substituent groups: a methyl group, an amino group, a carboxyl group, and a hydrogen atom. The two stereoisomers of alanine are nonsuperimposable mirror images of each other, and thus are enantiomers.

Compounds with asymmetric carbon atoms can be regarded as occurring in left- and right-handed forms, and are therefore called chiral compounds (Greek chiros, "hand"). Correspondingly, the asymmetric atom or center of chiral compounds is called the chiral atom or chiral center (Fig. 3–9). All but one of the 20 amino acids have chiral centers; glycine is the exception.

More generally, variations in the three-dimensional structure of biomolecules are described in terms of configuration and conformation. These terms are not synonyms. Configuration denotes the spatial arrangement of an organic molecule that is conferred by the presence of either (1) double bonds, around which there is no freedom of rotation, or (2) chiral centers, around which substituent groups are arranged in a specific sequence. The identifying characteristic of configurational isomers is that they cannot be interconverted without breaking one or more covalent bonds.

Figure 3–11a shows the configurations of maleic acid, which occurs in some plants, and its isomer fumaric acid, an intermediate in sugar metabolism. These compounds are geometric or cis–trans isomers; they differ in the arrangement of their substituent groups with respect to the nonrotating double bond. Maleic acid is the cis isomer and fumaric acid the trans isomer; each is a well-defined compound that can be isolated and purified. These two compounds are stereoisomers but not enantiomers; they are not mirror images of each other.

Molecular conformation refers to the spatial arrangement of substituent groups that are free to assume different positions in space, without breaking any bonds, because of the freedom of bond rotation. In the simple hydrocarbon ethane, for example, there is nearly complete freedom of rotation around the carbon–carbon single bond. Many different, interconvertible conformations of the ethane molecule are therefore possible, depending upon the degree of rotation (Fig. 3–12). Two conformations are of special interest: the staggered conformation, which is more stable than all others and thus predominates, and the eclipsed form, which is least stable. It is not possible to isolate either of these conformational forms, because they are freely interconvertible and in equilibrium with each other. However, when one or more of the hydrogen atoms on each carbon is replaced by a functional group that is either very large or electrically charged, freedom of rotation around the carbon–carbon single bond is hindered. This limits the number of stable conformations of the ethane derivative.
Figure 3–13 Stereoisomers that are distinguished by sensory receptors for smell and taste in humans.
(a) Two stereoisomers of carvone, designated R and S (see Fig. 3–10, legend). R-carvone (from spearmint oil) has the characteristic fragrance of spearmint; S-carvone (from caraway seed oil) smells like caraway.
(b) Aspartame, the artificial sweetener sold under the trade name NutraSweet, is easily distinguishable by taste from its bitter-tasting stereoisomer, although the two differ only in the configuration about one of the two chiral carbon atoms (in red).
Interactions between Biomolecules Are Stereospecific

Many biomolecules besides amino acids are chiral, containing one or more asymmetric carbon atoms. The chiral molecules in living organisms are usually present in only one of their chiral forms. For example, the amino acids occur in proteins only as the L isomers. Glucose, the monomeric subunit of starch, has five asymmetric carbons, but occurs biologically in only one of its chiral forms, the D isomer. (The conventions for naming stereoisomers of the amino acids are described in Chapter 5; those for sugars, in Chapter 11). In contrast, when a compound having an asymmetric carbon atom is chemically synthesized in the laboratory, the nonbiological reactions usually produce all possible chiral forms in an equimolar mixture that does not rotate polarized light (a racemic mixture). The chiral forms in such a mixture can be separated only by painstaking physical methods. Chiral compounds in living cells are produced in only one chiral form because the enzymes that synthesize them are also chiral molecules.

Stereospecificity, the ability to distinguish between stereoisomers, is a common property of enzymes and other proteins and a characteristic feature of the molecular logic of living cells. If the binding site on a protein is complementary to one isomer of a chiral compound, it will not be complementary to the other isomer, for the same reason that a left glove does not fit a right hand. Two striking examples of the ability of biological systems to distinguish stereoisomers are shown in Figure 3–13.

Chemical Reactivity

Saturated hydrocarbons – molecules with carbon–carbon single bonds and without double bonds or substituent groups – are not easily attacked by most chemical reagents; biomolecules, with their various functional groups, are much more chemically reactive. Functional groups alter the electron distribution and the geometry of neighboring atoms and thus affect the chemical reactivity of the entire molecule. The breakage and formation of chemical bonds during cellular metabolism release energy, some in the form of heat.

It is possible to analyze and predict the chemical behavior and reactions of biomolecules from the functional groups they bear. Enzymes recognize a specific pattern of functional groups in a biomolecule and catalyze characteristic chemical changes in the compound that contains these groups. Although a large number of different chemical reactions occur in a typical cell, these reactions are of only a few types, readily understandable in terms that apply to all reactions of organic compounds.

Bond Strength Is Related to the Electronegativities of the Bonded Atoms

When the two atoms sharing electrons in a covalent bond have equal affinities for the electrons, as in the case of two carbon atoms, the resulting bond is nonpolar. When two elements that differ in electron affinity, or electronegativity (Table 3–4), form a covalent bond (e.g., C and O), that bond is polarized; the shared electrons are more likely to be in the region of the more electronegative atom (O) than of the less electronegative (C). In the extreme case of two atoms of very different electronegativity (Na and Cl, for example), one of the atoms actually gives up the electron(s) to the other atom, resulting in the formation of ions and ionic interactions such as those in solid NaCl.

The strength of chemical bonds (Table 3–5) depends upon the relative electronegativities of the elements involved, the distance of the bonding electrons from each nucleus, and the nuclear charge. The number of electrons shared also influences bond strength; double bonds are stronger than single bonds, and triple bonds are stronger yet. The strength of a bond is expressed as bond energy, in joules. (In biochemistry, calories have often been used as units of energy – bond energy and free energy, for example. The joule is the unit of energy in the International System of Units, and is used throughout this book. For conversions, 1 cal is equal to 4.18 J.) Bond energy can be thought of as either the amount of energy required to break a bond or the amount of energy gained by the surroundings when two atoms form the bond. One way to put energy into a system is to heat it, which gives the molecules more kinetic energy; temperature is a measurement of the average kinetic energy of a population of molecules. When molecular motion is sufficiently violent, intramolecular vibrations and intermolecular collisions sometimes break chemical bonds. Heating raises the fraction of molecules with energies high enough to react.

In chemical reactions, bonds are broken and new ones are formed. The difference between the energy from the surroundings used to break bonds and the energy gained by the surroundings in the formation of new ones is virtually identical to the enthalpy change for the reaction, ΔH. (The energy difference becomes exactly equal to the enthalpy change after a slight correction for any volume change in the

system at constant pressure.) If heat energy is absorbed by the system as the change occurs (that is, if the reaction is endothermic), then ΔH has, by definition, a positive value; when heat is produced, as in exothermic reactions, ΔH is negative. In short, the change in enthalpy for a covalent reaction reflects the kinds and numbers of bonds that are made and broken. As we shall see later in this chapter, the enthalpy change is one of three factors that determine the free-energy change for a reaction; the other two are the temperature and the change in entropy.
Figure 3–14 Examples of five general types of chemical transformations that occur in cells. The reactions (a) through (d) are enzyme-catalyzed reactions that take place in your tissues as you use glucose as a source of energy (Chapter 14). In (a) a phosphoryl group is transferred from ATP to glucose; (b) an aldehyde is oxidized to a carboxylic acid and an oxidized electron carrier (NADP+) is reduced; (c) a rearrangement converts an aldehyde to a ketone; (d) a molecule is cleaved to form two smaller molecules. Reaction (e) represents the condensation of two amino acids with the elimination of H2O to form a peptide bond; condensation reactions occur in many cellular processes in which larger molecules are assembled from small precursors.
Five Types of Chemical Transformations Occur in Cells

Most cells have the capacity to carry out thousands of specific, enzyme-catalyzed reactions: transformation of simple nutrients such as glucose into amino acids, nucleotides, or lipids; extraction of energy from fuels by oxidation; or polymerization of subunits into macromolecules, for example. Fortunately for the student of biochemistry, there is a pattern in this multitude of reactions; we do not need to learn all of these reactions to comprehend the molecular logic of life.

Most of the reactions in living cells fall into one of five general categories (Fig. 3–14): functional-group transfers (a), oxidations and reductions (b), reactions that rearrange the bond structure around one or more carbons (c), reactions that form or break carbon–carbon bonds (d), and reactions in which two molecules condense, with the elimination of a molecule of water (e). Reactions within one category generally occur by similar mechanisms.

The mechanisms of biochemical reactions are not fundamentally different from other chemical reactions. Many biochemical reactions involve interactions between nucleophiles, functional groups rich in electrons and capable of donating them, and electrophiles, electrondeficient functional groups that seek electrons. Nucleophiles combine with, and give up electrons to, electrophiles. Functional groups containing oxygen, nitrogen, and sulfur are important biological nucleophiles (Table 3–6). Positively charged hydrogen atoms (protons) and positively charged metals (cations) frequently act as electrophiles in cells. A carbon atom can act as either a nucleophilic or an electrophilic center, depending upon which bonds and functional groups surround it.
Macromolecules and Their Monomeric Subunits

Many of the molecules found within cells are macromolecules, polymers of high molecular weight assembled from relatively simple precursors. Polysaccharides, proteins, and nucleic acids, which may have molecular weights ranging from tens of thousands to (in the case of DNA) billions, are produced by the polymerization of relatively small subunits with molecular weights of 500 or less. The synthesis of macromolecules is a major energy-consuming activity of cells. Macromolecules themselves may be further assembled into supramolecular complexes, forming functional units such as ribosomes, membranes, and organelles.

The Major Constituents of Organisms Are Macromolecules

Table 3–7 shows the major classes of biomolecules in a representative single-celled organism, Escherichia coli. Water is the most abundant single compound in E. coli and in all other cells and organisms. Inorganic salts and mineral elements, on the other hand, constitute only a very small fraction of the total dry weight, but many of them are in approximate proportion to their distribution in seawater (see Table 3–1). Nearly all of the solid matter in all kinds of cells is organic and is present in four forms: proteins, nucleic acids, polysaccharides, and lipids.

Proteins, long polymers of amino acids, constitute the largest fraction (besides water) of cells. Some proteins have catalytic activity and function as enzymes, others serve as structural elements, and still others carry specific signals (in the case of receptors) or specific substances (in the case of transport proteins) into or out of cells. Proteins are perhaps the most versatile of all biomolecules. The nucleic acids, DNA and RNA, are polymers of nucleotides. They store, transmit, and translate genetic information. The polysaccharides, polymers of simple sugars such as glucose, have two major functions: they serve as energy-yielding fuel stores and as extracellular structural elements. Shorter polymers of sugars (oligosaccharides) attached to proteins or lipids at the cell surface serve as specific cellular signals. The lipids, greasy or oily hydrocarbon derivatives, serve as structural components of membranes, as a storage form of energy-rich fuel, and in other roles. These four classes of large biomolecules are all synthesized in condensation reactions (Fig. 3–14e). In macromolecules – proteins, nucleic acids, and polysaccharides – the number of monomeric subunits is

very large. Proteins have molecular weights in the range of 5,000 to over 1 million; the nucleic acids have molecular weights ranging up to several billion; and polysaccharides, such as starch, also have molecular weights into the millions. Individual lipid molecules are much smaller (Mr 750 to 1,500), and are not classed as macromolecules. However, when large numbers of lipid molecules associate noncovalently, very large structures result. Cellular membranes are built of enormous aggregates containing millions of lipid molecules.
Figure 3–15 Informational and structural macromolecules. A, T, C, and G represent the four deoxynucleotides of DNA, and glucose (Glc) is the repeating monomeric subunit of starch and cellulose. The number of possible permutations and combinations of four deoxynucleotides is virtually limitless, as is the number of melodies possible with a few musical notes. A polymer of one subunit type is information-poor and monotonous.
Macromolecules Are Constructed from Monomeric Subunits

Although living organisms contain a very large number of different proteins and different nucleic acids, a fundamental simplicity underlies their structure (Chapter 1). The simple monomeric subunits from which all proteins and all nucleic acids are constructed are few in number and identical in all living species. Proteins and nucleic acids are informational macromolecules: each protein and each nucleic acid has a characteristic information-rich subunit sequence (Fig. 3–15).

Polysaccharides built from only a single kind of unit, or from two different alternating units, are not informational molecules in the same sense as are proteins and nucleic acids (Fig. 3–15). However, complex polysaccharides made up of six or more different kinds of sugars connected in branched chains do have the structural and stereochemical variety that enables them to carry information recognizable by other macromolecules.

Figure 3–16 The organic compounds from which most larger structures in cells are constructed: the ABCs of biochemistry. Shown on these two pages are (a) the 20 amino acids from which the proteins of all organisms are built (the side chains are shaded red), (b) the five nitrogenous bases, two five-carbon sugars, and phosphoric acid from which all nucleic acids are built, (c) five components found in many membrane lipids, and (d) α-D-glucose, the parent sugar from which most carbohydrates are derived. Note that phosphoric acid is a subunit of both nucleic acids and membrane lipids. The five-carbon and six-carbon sugars are shown here in their ring forms rather than their straightchain forms (Chapter 11). All components are shown in their un-ionized form.
Figure 3–17 Each simple component in Fig. 3–16 is a precursor of many other kinds of biomolecules.
Monomeric Subunits Have Simple Structures

Figure 3–16 shows the structures of some monomeric units, arranged in families. We have already seen that the most abundant polysaccharides in nature, starch and cellulose, are constructed of repeating units of D-glucose. The monomeric subunits of proteins are 20 different amino acids; all have an amino group (an imino group in the case of proline) and a carboxyl group attached to the same carbon atom, called, by convention, the α carbon. These α-amino acids differ from each other only in their side chains (Fig. 3–16).

The recurring structural units of all nucleic acids are eight different nucleotides; four kinds of nucleotides are the structural units of DNA, and four others are the units of RNA. Each nucleotide is made up of three components: (1) a nitrogenous organic base, (2) a five-carbon sugar, and (3) phosphate (Fig. 3–16). The eight different nucleotides of DNA and RNA are built from five different organic bases combined with two different sugars.

Lipids also are constructed from relatively few kinds of subunits. Most lipid molecules contain one or more long-chain fatty acids, of which palmitic acid and oleic acid are parent compounds. Many lipids also contain an alcohol, e.g., glycerol, and some contain phosphate (Fig. 3–16). Thus, only three dozen different organic compounds are the parents of most biomolecules.

Each of the compounds in Figure 3–16 has multiple functions in living organisms (Fig. 3–17). Amino acids are not only the monomeric subunits of proteins; some also act as neurotransmitters and as precursors of hormones and toxins. Adenine serves both as a subunit in the structure of nucleic acids and of ATP, and as a neurotransmitter. Fatty acids serve as components of complex membrane lipids, energy-rich fuel-storage fats, and the protective waxy coats on leaves and fruits. D-Glucose is the monomeric subunit of starch and cellulose, and also is the precursor of other sugars such as D-mannose and sucrose.

J. Willard Gibbs
Subunit Condensation Creates Order and Requires Energy

It is extremely improbable that amino acids in a mixture would spontaneously condense into a protein with a unique sequence. This would represent increased order in a population of molecules; but according to the second law of thermodynamics (Chapter 13) the tendency is toward ever-greater disorder in the universe. To bring about the synthesis of macromolecules from their monomeric subunits, free energy must be supplied to the system (the cell).

The randomness of the components of a chemical system is expressed as entropy, symbolized S. Any change in randomness of the system is the entropy change, ΔS, which has a positive value when randomness increases. J. Willard Gibbs, who developed the theory of energy changes during chemical reactions, showed that the free-energy content (G; recall Chapter 1) of any isolated system can be defined in terms of three quantities: enthalpy (H) (reflecting the number and kinds of bonds; see p. 66), entropy (S), and T, the absolute temperature (Kelvin). The definition of free energy is: G = HTS. When a chemical reaction occurs at constant temperature, the free-energy change is determined by ΔH, reflecting the kinds and numbers of chemical bonds and noncovalent interactions broken and formed, and ΔS, the change in the system’s randomness:


Recall from Chapter 1 that a process tends to occur spontaneously only if ΔG is negative. How, then, can cells synthesize polymers such as proteins and nucleic acids, if the free-energy change for polymerizing subunits is positive? They couple these thermodynamically unfavorable (endergonic) reactions to other cellular reactions that liberate free energy (exergonic reactions), so that the sum of the free-energy changes is negative:

Amino acids →  proteins ΔG1 is positive (endergonic)
ATP →  AMP + 2 PO43- ΔG2 is negative (exergonic)

Sum: Amino acids + ATP   →  proteins + AMP + 2 PO43-

The sum of ΔG1 and ΔG2 is negative (the overall process is exergonic).
Figure 3–18 The structural hierarchy in the molecular organization of cells. The nucleus of this plant cell, for example, contains several types of supramolecular complexes, including chromosomes. Chromosomes consist of macromolecules – DNA and many different proteins. Each type of macromolecule is constructed from simple subunits – DNA from the deoxyribonucleotides, for example. (Adapted from Becker, W.M. and Deamer, D.W. (1991) The World of the Cell, 2nd edn, Fig. 2–15, The Benjamin/Cummings Publishing Company, Menlo Park, CA)
There Is a Hierarchy in Cell Structure

The monomeric subunits in Figure 3–16 are very small compared with biological macromolecules. An amino acid molecule such as alanine is less than 0.5 nm long. Hemoglobin, the oxygen-carrying protein of erythrocytes, consists of nearly 600 amino acid units covalently linked into four long chains, which are folded into globular shapes and associated in a tetrameric structure with a diameter of 5.5 nm. Protein molecules in turn are small compared with ribosomes (about 20 nm in diameter), which contain about 70 different proteins and several different RNA molecules. Ribosomes, in their turn, are much smaller than organelles such as mitochondria, typically 1,000 nm in diameter. It is a long jump from the simple biomolecules to the larger cellular structures that can be seen with the light microscope. Figure 3–18 illustrates the structural hierarchy in cellular organization.

In proteins, nucleic acids, and polysaccharides, the individual subunits are joined by covalent bonds. By contrast, in supramolecular complexes, the different macromolecules are held together by noncovalent interactions – much weaker, individually, than covalent bonds. Among these are hydrogen bonds (between polar groups), ionic interactions (between charged groups), hydrophobic interactions (between nonpolar groups), and van der Waals interactions, all of which have energies of only a few kilojoules, compared with covalent bonds, which have bond energies of 200 to 900 kJ/mol (see Table 3–5). The nature of these noncovalent interactions will be described in the next chapter.

The large numbers of weak interactions between macromolecules in supramolecular complexes stabilize the resulting noncovalent structures.

Although the monomeric subunits of macromolecules are so much smaller than cells and organelles, they influence the shape and function of these much larger structures. In sickle-cell anemia, a hereditary human disorder, the hemoglobin molecule is defective. In the two β chains of hemoglobin from healthy individuals, a glutamic acid residue occurs at position 6. In people with sickle-cell anemia, a valine residue occurs at position 6. This single difference in the sequence of the 146 amino acids of the β chain affects only a tiny portion of the molecule, yet it causes the hemoglobin to form large aggregates within the erythrocytes, which become deformed (sickled) and function abnormally.

Prebiotic Evolution

Because all biological macromolecules are made from the same three dozen subunits, it seems likely that all living organisms descended from a single primordial cell line. These subunits are proposed to have had, singly and collectively, the most successful combination of chemical and physical properties for their function as the raw materials of biological macromolecules and for carrying out the basic energy-transforming and self-replicating features of a living cell. These primordial organic compounds may have been retained during biological evolution over billions of years because of their unique fitness.

Figure 3–19 Lightning evoked by a volcanic eruption that resulted in the formation of the island of Surtsey off the coast of Iceland in 1963. The intense fields of electrical, thermal, and shock-wave energy generated by such cataclysms, which were frequent on the primitive earth, could have been a major factor in the origin of organic compounds.
Biomolecules First Arose by Chemical Evolution

We come now to a puzzle. Apart from their occurrence in living organisms, organic compounds, including the basic biomolecules, occur only in trace amounts in the earth’s crust, the sea, and the atmosphere. How did the first living organisms acquire their characteristic organic building blocks? In 1922, the biochemist Aleksandr I. Oparin proposed a theory for the origin of life early in the history of the earth, postulating that the atmosphere was once very different from that of today. Rich in methane, ammonia, and water, and essentially devoid of oxygen, it was a reducing atmosphere, in contrast to the oxidizing environment of our era. In Oparin’s theory, electrical energy of lightning discharges or heat energy from volcanoes (Fig. 3–19) caused ammonia, methane, water vapor, and other components of the primitive atmosphere to react, forming simple organic compounds. These compounds then dissolved in the ancient seas, which over many millenia became enriched with a large variety of simple organic compounds. In this warm solution (the "primordial soup") some organic molecules had a greater tendency than others to associate into larger complexes. Over millions of years, these in turn assembled spontaneously to form membranes and catalysts (enzymes), which came together to become precursors of the first primitive cells. For many years, Oparin’s views remained speculative and appeared untestable.

Figure 3–20 Spark-discharge apparatus of the type used by Miller and Urey in experiments demonstrating abiotic formation of organic compounds under primitive atmospheric conditions. After subjecting the gaseous contents of the system to electrical sparks, products were collected by condensation. Biomolecules such as amino acids were among the products (see Table 3–8).
Figure 3–21 Among the products of electrical discharge through an atmosphere containing HCN are compounds such as those in (a). These compounds promote the polymerization of monomers such as amino acids into polymers (b).
Chemical Evolution Can Be Simulated in the Laboratory

A classic experiment on the abiotic (nonbiological) origin of organic biomolecules was carried out in 1953 by Stanley Miller in the laboratory of Harold Urey. Miller subjected gaseous mixtures of NH3, CH4, water vapor, and H2 to electrical sparks produced across a pair of electrodes (to simulate lightning) for periods of a week or more (Fig. 3–20), then analyzed the contents of the closed reaction vessel. The gas phase of the resulting mixture contained CO and CO2, as well as the starting

materials. The water phase contained a variety of organic compounds, including some amino acids, hydroxy acids, aldehydes, and hydrogen cyanide (HCN). This experiment established the possibility of abiotic production of biomolecules in relatively short times under relatively mild conditions.

Several developments have allowed more refined studies of the type pioneered by Miller and Urey, and have yielded strong evidence that a wide variety of biomolecules, including proteins and nucleic acids, could have been produced spontaneously from simple starting materials probably present on the earth at the time life arose.

Modern extensions of the Miller experiments have employed "atmospheres" that include CO2 and HCN, and much improved technology for identifying small quantities of products. The formation of hundreds of organic compounds has been demonstrated (Table 3–8). These compounds include more than ten of the common amino acids, a variety of mono-, di-, and tricarboxylic acids, fatty acids, adenine, and formaldehyde. Under certain conditions, formaldehyde polymerizes to form sugars containing three, four, five, and six carbons. The sources of energy that are effective in bringing about the formation of these compounds include heat, visible and ultraviolet (UV) light, x rays, gamma radiation, ultrasound and shock waves, and alpha and beta particles.

In addition to the many monomers that form in these experiments, polymers of nucleotides (nucleic acids) and of amino acids (proteins) also form. Some of the products of the self condensation of HCN are effective promoters of such polymerization reactions (Fig. 3–21), and inorganic ions present in the earth’s crust (Cu2+, Ni2+, and Zn2+) also enhance the rate of polymerization.

In short, laboratory experiments on the spontaneous formation of biomolecules under prebiotic conditions have provided good evidence that many of the chemical components of living cells, including proteins and RNA, can form under these conditions. Short polymers of RNA can act as catalysts in biologically significant reactions (Chapter 25), and it seems likely that RNA played a crucial role in prebiotic evolution, both as catalyst and as information repository.

Figure 3–22 One possible "RNA world" scenario, showing the transition from the prebiotic RNA world (shades of yellow) to the biotic DNA world (orange).
RNA Molecules May Have Been the First Genes and Catalysts

In modern organisms, nucleic acids encode the genetic information that specifies the structure of enzymes, and enzymes have the ability to catalyze the replication and repair of nucleic acids. The mutual dependence of these two classes of biomolecules poses the perplexing question: which came first, DNA or protein?

The answer may be: neither. The discovery that RNA molecules can act as catalysts in their own formation suggests that RNA may have been the first gene and the first catalyst. According to this scenario (Fig. 3–22), one of the earliest stages of biological evolution was the chance formation, in the primordial soup, of an RNA molecule that had the ability to catalyze the formation of other RNA molecules of the same sequence – a self-replicating, self-perpetuating RNA. The concentration of a self-replicating RNA molecule would increase exponentially, as one molecule formed two, two formed four, and so on. The fidelity of self replication was presumably less than perfect, so the process would generate variants of the RNA, some of which might be even better able to self-replicate. In the competition for nucleotides, the most efficient of the self-replicating sequences would win, and less efficient replicators would fade from the population.

The division of function between DNA (genetic information storage) and protein (catalysis) was, according to the "RNA world" hypothesis, a later development (Fig. 3–22). New variants of self-replicating RNA molecules developed, with the additional ability to catalyze the condensation of amino acids into peptides. Occasionally, the peptide(s) thus formed would reinforce the self-replicating ability of the RNA, and the pair – RNA molecule and helping peptide – could undergo further modifications in sequence, generating even more efficient self-replicating systems. Sometime after the evolution of this primitive protein-synthesizing system, there was a further development: DNA molecules with sequences complementary to the self-replicating RNA molecules took over the function of conserving the "genetic" information, and RNA molecules evolved to play roles in protein synthesis. Proteins proved to be versatile catalysts, and over time, assumed that function. Lipidlike compounds in the primordial soup formed relatively impermeable layers surrounding self-replicating collections of molecules. The concentration of proteins and nucleic acids within these lipid enclosures favored the molecular interactions required in self-replication.

This "RNA world" hypothesis is plausible but by no means universally accepted. The hypothesis does make testable predictions, and to the extent that experimental tests are possible within finite times (less than or equal to the life span of a scientist!), the hypothesis will be tested and refined.

Figure 3–23 Ancient reefs in Australia contain fossil evidence of microbial life in the sea of 3.5 billion years ago. Bits of sand and limestone became trapped in the sticky extracellular coats of cyanobacteria, gradually building up these stromatolites found in Hamelin Bay, Western Australia (a). Microscopic examination of thin sections of stromatolite reveals microfossils of filamentous bacteria (b).
Biological Evolution Began More Than Three Billion Years Ago

The earth was formed about 4.5 billion years ago, and the first definitive evidence of life dates to about 3.5 billion years ago. An international group of scientists showed in 1980 that certain ancient rock formations (stromatolites; Fig. 3–23) in western Australia contained fossils of primitive microorganisms. Somewhere on earth during that first billion-year period, there arose the first simple organism, capable

of replicating its own structure from a template (RNA?) that was the first genetic material. Because the terrestrial atmosphere at the dawn of life was nearly devoid of oxygen, and because there were few microorganisms to scavenge organic compounds formed by natural processes, these compounds were relatively stable. Given this stability and eons of time, the improbable became inevitable: the organic compounds were incorporated into evolving cells to produce more and more effective self reproducing catalysts. The process of biological evolution had begun. Organisms developed mechanisms for harnessing the energy of sunlight through photosynthesis, to make sugars and other organic molecules from carbon dioxide, and to convert molecular nitrogen from the atmosphere into nitrogenous biomolecules such as amino acids. By developing their own capacities to synthesize biomolecules, cells became independent of the random processes by which such compounds had first appeared on earth. As evolution proceeded, organisms began to interact and to derive mutual benefits from each other’s products, forming increasingly complex ecological systems.
    3.S:         3.X:      3.P:  Chapter 4:         4.1:            4.1.1:            4.1.2:            4.1.3:            4.1.4:            4.1.5:            4.1.6:            4.1.7:            4.1.8:      4.2:            4.2.1:            4.2.2:            BOX 4–1:            4.2.3:            4.2.4:            4.2.5:      4.3:            4.3.1:            4.3.2:            BOX 4–2:            4.3.3:            4.3.4:            BOX 4–3:      4.4:      4.5:      4.S:      4.X:      4.P:  Part II:     Chapter 5:         5.1:            5.1.1:            5.1.2:            5.1.3:            5.1.4:            BOX 5–1:            5.1.5:            5.1.6:            5.1.7:            5.1.8:            5.1.9:            5.1.10:            5.1.11:      5.2:            5.2.1:            5.2.2:            5.2.3:            5.2.4:            BOX 5–2:      5.S:      5.X:      5.P:  Chapter 6:         6.1:            6.1.1:            6.1.2:            6.1.3:            6.1.4:      6.2:            6.2.1:            6.2.2:            6.2.3:            6.2.4:      6.3:            6.3.1:            6.3.2:            6.3.3:            6.3.4:            6.3.5:            6.3.6:            6.3.7:      6.S:      6.X:      6.P:  Chapter 7:         7.1:            7.1.1:            7.1.2:      7.2:            7.2.1:            7.2.2:            BOX 7–1:            7.2.3:            7.2.4:            7.2.5:            7.2.6:            7.2.7:            BOX 7–2:      7.3:            7.3.1:            BOX 7–3:            7.3.2:            7.3.3:            7.3.4:            7.3.5:            7.3.6:            7.3.7:      7.4:            7.4.1:            7.4.2:            7.4.3:            7.4.4:            7.4.5:      7.S:      7.X:      7.P:  Chapter 8:         8.1:            8.1.1:            8.1.2:      8.2:            8.2.1:            8.2.2:            8.2.3:            8.2.4:            8.2.5:            8.2.6:      8.3:            8.3.1:            8.3.2:            BOX 8–1:            8.3.3:            8.3.4:            8.3.5:            8.3.6:            BOX 8–2:            8.3.7:      8.4:            8.4.1:            BOX 8–3:            99999:      *.*: