Foundations of Biochemistry I

xx part

1 The Molecular Logic of Life facing page 2 Cells The Orion Nebula, a tremendous cloud of gas in which many hot, young stars are evolving rapidly toward cata- 3 Biomolecules clysmic cosmic explosions called supernovae. Energy released by nuclear explosions in such supernovae 4 Water brought about the fusion of simple atomic nuclei, forming the more complex elements of which the earth, Fifteen to twenty billion years ago, the universe arose as a cataclysmic its atmosphere, and all living things are composed. eruption of hot, energy-rich subatomic particles. Within seconds, the simplest elements (hydrogen and helium) were formed. As the universe ex- panded and cooled, material condensed under the influence of gravity to form stars. Some stars became enormous and then 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 earth itself and the chemical elements found on the earth today. About four billion years ago, life arose—simple microorganisms with the ability to extract energy from organic compounds or from sunlight, which they used to make a vast array of more complex biomolecules from the simple elements and compounds on the earth’s surface. Biochemistry asks how the thousands of different biomolecules 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 obey 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 chemical transformations, assemble complex molecules from simpler subunits, form supramolecular structures 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 have led to such diversity. Nearly all of the organic compounds from which living organisms are constructed are products of biological activity. These molecules were se- lected during the course of biological evolution for their fitness in perform- ing specific biochemical and cellular functions. Biomolecules can be char- acterized and understood in the same terms that apply to molecules of inanimate matter: the types of bonds between atoms, the factors that con- tribute to bond formation and bond strength, the three-dimensional

1 2 Part I Foundations of Biochemistry

structures of molecules, and chemical reactivities. Three-dimensional structure is especially important in biochemistry. Biological interactions, such as those between enzyme and substrate, antibody and antigen, hor- mone and receptor, are highly specific, and this specificity is achieved by steric and electrostatic complementarity between molecules. Prominent among the forces that stabilize three-dimensional structure are noncovalent interactions, individually weak but with significant cumulative effects. 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 it is the solvent in which most biochemical transformations occur. The properties of water have shaped the course of evolution, and the structure and interactions of biomolecules are profoundly influenced by the aqueous solution in which biomolecules reside. 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 whatever your background in chemistry or biology, you can immediately begin to fol- low, and to enjoy, the action. chapter

The Molecular Logic of Life 1

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 exhibited 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, and then we describe a set of principles that characterize all living organisms. These principles underlie the organization of organisms and their cells, 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. (a)

The Chemical Unity of Diverse Living Organisms What distinguishes living organisms from inanimate objects? First is their degree of chemical complexity and organization. Thousands of different molecules make up a cell’s intricate internal structures (Fig. 1–1a). By contrast, inanimate matter—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 chemical nutrients or sun- (b) light. This energy enables organisms to build and maintain their intricate structures and to do mechanical, chemical, osmotic, and other types of work. Inanimate matter does not use energy in a systematic, dynamic way to maintain structure or to do work; rather, it tends to decay toward a more disordered state, to come to equilibrium with its surroundings. The third attribute of living organisms is the capacity for precise self- replication and self-assembly, a property that is the quintessence of the living state (Fig. 1–1c). 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, its construction directed entirely from information contained within the genetic material of (c) the original cell. Although the ability to self-replicate has no true analog in the nonliving figure 1–1 Some characteristics of living matter. (a) Microscopic world, there is an instructive analogy in the growth of crystals in saturated complexity and organization are apparent in this colorized solutions. Crystallization produces more material identical in lattice struc- thin section of vertebrate muscle tissue, viewed with the ture to the original “seed” crystal. Crystals are much less complex than the electron microscope. (b) A prairie falcon acquires nutri- simplest living organisms, and their structure is static, not dynamic as are ents by consuming a smaller bird. (c) Biological repro- living cells. Nevertheless, the ability of crystals to “reproduce” themselves duction occurs with near-perfect ﬁdelity.

3 4 Part I Foundations of Biochemistry

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 our modern understanding of gene structure) 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 nu- cleus or chloroplast and of individual chemical compounds. The interplay among the chemical components of a living organism is dynamic; changes in one component cause coordinating or compensating changes in another, Erwin Schrödinger with the whole ensemble displaying a character beyond that of its individ- 1887–1961 ual constituents. The collection of molecules carries out a program, the end result of which is reproduction of the program and self-perpetuation of that collection of molecules; in short, life.

Biochemistry Explains Diverse Forms of Life in Unifying Chemical Terms 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 can a living organism 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, called vi- talism, has been ﬁrmly rejected by modern science. The study of biochemistry shows how the collections of inanimate molecules that constitute living organisms interact to maintain and perpetuate life animated solely by the chemical laws that govern the nonliving universe. Living organisms are enormously diverse (Fig. 1–2). In appearance and function, birds and beasts, trees, grasses, and microscopic organisms differ

figure 1–2 Diverse living organisms share common chemical features. Birds, beasts, plants, and soil microorganisms share with humans the same basic structural units (cells) and the same kinds of macromolecules (DNA, RNA, proteins) made up of the same kinds of monomeric subunits (nucleotides, amino acids). They utilize the same pathways for synthesis of cellular components, share the same genetic code, and derive from the same evolutionary ancestors. (“The Garden of Eden” (detail), by Jan van Kessel, the Younger (1626–1679).) Chapter 1 The Molecular Logic of Life 5

greatly. Yet, biochemical research has revealed that all organisms are re- Monomeric subunits markably alike at the cellular and chemical levels. Biochemistry describes Letters of Deoxyribo- Amino in molecular terms the structures, mechanisms, and chemical processes English nucleotides acids shared by all organisms, and provides organizing principles that underlie life alphabet (26 different (4 different (20 different in all of its diverse forms, principles we shall refer to collectively as the mol- kinds) kinds) kinds) ecular logic of life. Although biochemistry provides important insights and practical applications in medicine, agriculture, nutrition, and industry, its Thr J T R Phe Tyr ultimate concern is with the wonder of life itself. H D C Gly Asn Gln Despite the fundamental unity of life, very few generalizations about Z Y N Asp Q Lys Met living organisms are absolutely correct for every organism under every con- B V FI A Ala Ser dition. The range of habitats in which organisms live, from hot springs to U S Val A Arg P T Ile Arctic tundra, from animal intestines to college dormitories, is matched by O His W E M Leu a correspondingly wide range of speciﬁc biochemical adaptations, achieved Pro G X Glu within a common chemical framework. For the sake of clarity, we will some- K L C G Trp Cys times risk certain generalizations, which, though not perfect, remain useful; we will also frequently point out the exceptions that illuminate scientiﬁc generalizations.

MM P G A G Gly Arg Asp

All Macromolecules Are Constructed from a Few Simple Compounds EAA A G A Val Gly Gly Most of the molecular constituents of living systems are composed of car- Asp Pro Pro bon atoms covalently joined with other carbon atoms and with hydrogen, SSS T T C oxygen, or nitrogen. The special bonding properties of carbon permit the SSS C C T Ser Leu Leu formation of a great variety of molecules. Organic compounds of molecular 1 AAA A T A Phe Lys Gly weight (also called relative molecular mass, Mr) less than about 500, such as amino acids, nucleotides, and monosaccharides, serve as monomeric GGG T T C Arg Asn Lys subunits of macromolecules: proteins, nucleic acids, and polysaccharides. A single protein molecule may have 1,000 or more amino acids, and EEE A G G Glu Phe Trp deoxyribonucleic acid has millions of nucleotides. SSS C C T Ala Glu Cys Each cell of the bacterium Escherichia coli (E. coli) contains several thousand kinds of organic compounds, including a thousand different pro- English Deoxyribonucleic Protein teins, a similar number of different nucleic acid molecules, and hundreds of words acid (DNA) types of carbohydrates and lipids. In humans there may be tens of thou- Ordered linear sequences sands of different proteins, as well as many types of polysaccharides (chains of simple sugars), a variety of lipids, and many other compounds of lower molecular weight. For a segment of 8 subunits, the number of To purify and to characterize thoroughly all of these molecules would different sequences possible = be an insuperable task were it not for the fact that each class of macromol- 268 or 48 or 208 or ecules (proteins, nucleic acids, polysaccharides) is composed of a small, 2.1 1011 65,536 2.56 1010 common set of monomeric subunits. These monomeric subunits can be covalently linked in a virtually limitless variety of sequences (Fig. 1–3), just figure 1–3 as the 26 letters of the English alphabet can be arranged into a limitless Monomeric subunits in linear sequences can spell inﬁ- number of words, sentences, and books. nitely complex messages. The number of different sequences possible (S) depends on the number of dif- Deoxyribonucleic acids (DNA) are constructed from only four different kinds of subunits (N ) and the length of the linear ferent kinds of simple monomeric subunits, the deoxyribonucleotides. sequence (L): S NL. For an average-sized protein Ribonucleic acids (RNA) are composed of just four types of ribonu- (L 400), S is 20400—an astronomical number. cleotides. Proteins are composed of 20 different kinds of amino acids. The eight nucleotides from which all nucleic acids are built and the 20 different amino acids from which all proteins are built are identical in all living

1The terms used to indicate the size of a molecule are often confused. We use molecular weight or Mr , relative molecular mass, a dimensionless ratio of the mass of a molecule to one-twelfth the mass of 12C. The size of a molecule can also be correctly given in terms of molecular mass (m), which has units of daltons (Da) or atomic mass units (amu). A molecule should never be described as having a molecular weight or Mr (a dimensionless property) expressed in daltons or atomic mass units. 6 Part I Foundations of Biochemistry

organisms. The speciﬁc sequence of monomeric subunits together with their arrangement in space shapes macromolecules for their particular biological functions as genes, catalysts, hormones, and so on. Most of the monomeric subunits from which all macromolecules are constructed serve more than one function in living cells. Nucleotides serve not only as subunits of nucleic acids but also as energy-carrying molecules. Amino acids are subunits of protein molecules and are also precursors of hormones, neurotransmitters, pigments, and many other kinds of biomolecules. We can now set out some of the principles in the molecular logic of life:

All living organisms build molecules from the same kinds of monomeric subunits. The structure of a macromolecule determines its speciﬁc biological function. Each genus and species is deﬁned by its distinctive set of macromolecules.

Energy Production and Consumption in Metabolism Energy is a central theme in biochemistry: cells and organisms depend on a constant supply of energy to oppose the inexorable tendency in nature for a system to decay to its lowest energy state. The storage and expression of information costs energy, without which structures rich in information in-

evitably become disordered and meaningless. The synthetic reactions that [K ]fish [K ]lake [Na ]fish [Na ]lake occur within cells, like the synthetic processes in any factory, require the in- [Cl ]fish [Cl ]lake put 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. Cells have evolved highly efficient mechanisms for coupling the energy [K ]body [K ]lake obtained from sunlight or fuels to the many energy-consuming processes [Na ]body [Na ]lake Phyto- [Cl] [Cl] they carry out. plankton body lake

2 HPO4 Organisms Are Never at Equilibrium with Their Surroundings CO2 One of the first developments in biological evolution must have been an oily NH 3 membrane that enclosed the water-soluble molecules of the primitive cell, K Na segregating them and allowing them to accumulate to relatively high con- Monomeric Cl subunits centrations. The molecules and ions contained within a living organism differ in kind and in concentration from those in the organism’s surroundings. For example, the cells of a freshwater fish contain certain inorganic ions at DNA, RNA, protein, lipids, etc. concentrations far different from those in the surrounding water (Fig. 1–4). Proteins, nucleic acids, sugars, and fats are present in the fish but are essen- tially absent from the surrounding medium, which contains only simpler mol- figure 1–4 ecules such as carbon dioxide, molecular oxygen, and water. Only by contin- Living organisms are not at equilibrium with their sur- uously expending energy can the fish establish and maintain its constituents roundings. Death and decay restore the equilibrium. at concentrations distinct from those of the surroundings. When the fish During life, the fish uses energy from food to build dies, its components eventually come to equilibrium with its surroundings. complex molecules and to concentrate ions from the surroundings. When it dies, it no longer derives energy from food and thus cannot maintain concentration gradients; Molecular Composition Reflects a Dynamic Steady State ions leak out. Inexorably, macromolecular components Although the chemical composition of an organism may be almost constant decay to simpler compounds. These simple compounds through time, the population of molecules within a cell or organism is far serve as nutritional sources for microscopic plants and algae (the phytoplankton), which are then eaten by larger from static. Molecules are synthesized and then broken down by continuous organisms. (By convention, square brackets denote con- chemical reactions, involving a constant flux of mass and energy through centration—in this case, of ionic species.) the system. The hemoglobin molecules carrying oxygen from your lungs to Chapter 1 The Molecular Logic of Life 7

synthesis degradation Precursors Hemoglobin Breakdown products figure 1–5 (amino acids)r1 (in erythrocyte)r2 (amino acids) The dynamic steady state. A dynamic steady state results when the rate of appearance of a cellular component is exactly matched by the rate of its disappearance. When r1 r2, the concentration of hemoglobin is constant. In this scheme, r1, r2, and so forth, represent the rates of (a) the various processes. 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 r2 Waste CO2 be consumed by metabolic processes in other tissues ingestion utilization r3 (heart, brain, skeletal muscle). The dynamic steady-state Food Glucose Storage fats (carbohydrates)r1 (in blood) concentrations of hemoglobin and glucose are maintained r 4 Other products by complex mechanisms regulating the relative rates of the processes shown here.

When r1 r2 r3 r4, the concentration of glucose in blood is constant. (b)

your brain at this moment were synthesized within the past month; by next month they will have been degraded and replaced by 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 amounts of hemoglobin and glucose in the blood remain 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 is the result of a dynamic steady state.

Organisms Transform Energy and Matter from 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—the energy transformations and exchanges on which all living organisms depend. For chemical reactions occurring in solution, we can define a system as all of the reactants and products present, 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. If the system exchanges energy but not matter with its surroundings, it is an isolated system; if it exchanges both energy and material with its surroundings, it is an open system. 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 energy from their surroundings: (1) they take up chemical fuels from the environment and extract energy by oxidizing them; or (2) they absorb energy from sunlight.

Living organisms create and maintain their complex, orderly structures using energy extracted from fuels or sunlight. 8 Part I Foundations of Biochemistry

• Nutrients in environment figure 1–6 (complex molecules such as During metabolic transductions, the randomness of the Potential energy sugars, fats) system plus surroundings (expressed quantitatively as • Sunlight entropy) increases as the potential energy of complex nutrient molecules decreases. Living organisms (a) extract (a) energy from their environment; (b) convert some of it into useful forms of energy to produce work; (c) return some energy to the environment as heat; and (d) release end- Chemical transformations product molecules that are less well organized than the within cells starting fuel, increasing the entropy of the universe. One effect of all these transformations is (e) increased order Energy (decreased randomness) in the form of complex macro- transductions Cellular work: molecules. We shall return to a quantitative treatment of accomplish • chemical synthesis work • mechanical work entropy in Chapter 14. • osmotic and electrical gradients • light production • genetic information transfer The first law of thermodynamics, developed from physics and chem- (b) istry but fully valid for biological systems as well, describes the principle of the conservation of energy: Heat In any physical or chemical change, the total amount of energy in (c) the universe remains constant, although the form of the energy may change. Increased randomness (entropy) in the surroundings Cells are consummate transducers of energy, capable of interconverting Metabolism produces compounds chemical, electromagnetic, mechanical, and osmotic energy with great effi- simpler than the initial ciency (Fig. 1–6). Biological energy transducers differ from many familiar fuel molecules: CO2, NH3, machines that depend on temperature or pressure differences. The steam H O, HPO2 2 4 engine, for example, converts the chemical energy of fuel into heat, raising (d) the temperature of water to its boiling point to produce steam pressure that drives a mechanical device. The internal combustion engine, similarly, de- Decreased randomness pends upon changes in temperature and pressure. By contrast, all parts of (entropy) in the system a living organism must operate at about the same temperature and pres- Simple compounds polymerize sure, and heat flow is therefore not a useful source of energy. to form information-rich macromolecules: DNA, RNA, Living cells are chemical engines that function at constant temper- proteins ature.

(e) The Flow of Electrons Provides Energy for Organisms Nearly all living organisms derive their energy, directly or indirectly, from the radiant energy of sunlight, which arises from thermonuclear fusion reactions occurring in the sun (Fig. 1–7). Photosynthetic cells absorb light energy and use it to drive electrons from water to carbon dioxide, forming energy-rich products such as starch and sucrose and releasing molecular oxygen into the atmosphere (Fig. 1–8). Nonphotosynthetic cells and organisms obtain the energy they need by oxidizing the energy-rich products of photosynthesis and then passing electrons to atmospheric oxygen to 4H form water, carbon dioxide, and other end products, which are recycled in Thermonuclear fusion the environment. Virtually all energy transductions in cells can be traced to 4He

figure 1–7 Sunlight is the ultimate source of all biological energy. Thermonuclear reactions in the sun produce helium from hydrogen and release electromagnetic energy, which is transmitted to the earth as light and converted Photons of into chemical energy by plants and some algae and visible light bacteria. Chapter 1 The Molecular Logic of Life 9 this flow of electrons from one molecule to another, in a “downhill” flow Photosynthesis in plants, from higher to lower electrochemical potential; as such, it is formally anal- algae, bacteria ogous to the flow of electrons in a battery-driven electric circuit. All these reactions involving electron flow are oxidation-reduction reactions; some reactant is oxidized (loses electrons) as another is reduced (gains electrons).

CO2 Reduced fuels The energy needs of virtually all organisms are provided, directly and O2 or indirectly, by solar energy. The ﬂow of electrons in oxidation-reduction reactions underlies energy transductions in living cells. Cellular respiration in Living organisms are interdependent, exchanging energy and mat- animals, plants, algae, bacteria ter via the environment. figure 1–8 Energy Coupling Links Reactions in Biology Photosynthetic organisms (plants, some algae, and some bacteria) are the ultimate providers of fuels—reduced, The central issue in bioenergetics is the means by which energy from fuel energy-rich compounds—in the biosphere. The energy metabolism or light capture is coupled to energy-requiring reactions. It is of sunlight drives the synthesis of fuels such as sucrose instructive to consider the simple mechanical example of energy coupling and starch, with O2 as a by-product. These fuels, or the shown in Figure 1–9a. An object at the top of an inclined plane has a cer- photosynthetic organisms themselves, are then a source tain amount of potential energy as a result of its elevation. It tends sponta- of food for animals, which oxidize the sucrose and starch (using O2 and producing CO2) to supply energy. This neously to slide down the plane, losing its potential energy of position as it process of fuel oxidation—cellular respiration—is the approaches the ground. When an appropriate string-and-pulley device cou- energy source for metabolism in both photosynthetic and ples the falling object to another, smaller object, the spontaneous down- nonphotosynthetic organisms. ward motion of the larger can lift the smaller, accomplishing a certain amount of work. The amount of energy actually available to do work, called the free energy, G, will always be somewhat less than the theoretical amount of energy released, because some energy is dissipated as the heat of friction. The greater the elevation of the larger object relative to its ﬁnal (a) Mechanical example position, the greater is the release of energy as it slides downward, and the greater the amount of work that can be accomplished. ∆G > 0 ∆G < 0 Chemical reactions can also be coupled so that an energy-releasing reaction drives an energy-requiring one. Chemical reactions in closed systems Work Loss of proceed spontaneously until equilibrium is reached. When a system is at done potential raising energy of equilibrium, the rate of product formation exactly equals the rate at which object position

figure 1–9 Energy coupling in mechanical and chemical processes. Endergonic Exergonic (a) The downward motion of an object releases potential energy that can do mechanical work. The potential energy made available by spontaneous downward motion, an (b) Chemical example exergonic process (pink), can be coupled to the endergonic upward movement of another object (blue). (b) In reac- Reaction 2: → tion 1, the formation of glucose 6-phosphate from glucose ATP ADP Pi Reaction 3: Glucose ATP → and inorganic phosphate, Pi, yields a product of higher glucose 6-phosphate ADP energy than the two reactants. For this endergonic reac- G tion, G is positive. In reaction 2, the exergonic break- Reaction 1: → Glucose Pi down of adenosine triphosphate (ATP; see Fig. 1–10) can glucose 6-phosphate drive an endergonic reaction when the two reactions are ∆G ∆ 2 G3 coupled. The exergonic reaction has a large, negative free-energy change (G ), and the endergonic reaction 2 Free energy, ∆G has a smaller, positive free-energy change (G1). The 1 ∆ ∆ ∆ third reaction accomplishes the sum of reactions 1 and 2, G3 = G1 G2

and the free-energy change, G3, is the arithmetic sum of G1 and G2. Because the value of G3 is negative, the overall reaction is exergonic and proceeds spontaneously. Reaction coordinate 10 Part I Foundations of Biochemistry

product is converted to reactant. Thus there is no net change in the concentration of reactants and products; a “steady state” is achieved. The energy change as the system moves from its initial state to equilibrium, with no changes in temperature or pressure, is given by the free-energy change, G. The magnitude of G depends on the particular chemical reaction and on how far from equilibrium the system is initially. Each compound involved in a chemical reaction contains a certain amount of potential energy, related to the kind and number of its bonds. In reactions that occur spontaneously, the products have less free energy than the reactants, thus the reaction releases free energy, which is then available to do work. Such reactions are exergonic; the decline in free energy from reactants to products is expressed as a negative value. Endergonic reactions require an input of energy, and their G values are therefore positive. As in mechanical processes, only part of the energy released in exergonic biochemical reactions can be used to accomplish work. In living systems some energy is dissipated as heat or lost to increasing entropy, a measure of randomness, which we will deﬁne more rigorously in Chapter 14. In living organisms, as in the mechanical example in Figure 1–9a, an exergonic reaction can be coupled to an endergonic reaction or process to drive otherwise unfavorable reactions. Figure 1–9b illustrates this principle for the case of glucose 6-phosphate synthesis, a reaction occurring in muscle cells. The simplest way to produce glucose 6-phosphate would be reac-

tion 1, which is endergonic. (Pi is an abbreviation for inorganic phosphate, 2 HPO4 . Don’t be concerned about the structure of these compounds now; we will describe them in detail later.) Reaction 1: Glucose Pi 88n glucose 6-phosphate (endergonic, G is positive) In this reaction, the product contains more energy than the reactants. A second, very exergonic reaction can occur in living cells. Reaction 2: ATP 88n ADP Pi (exergonic, G is negative) In this reaction, the products contain less energy than the reactant—the reaction releases energy. The two chemical reactions share a common inter-

mediate, Pi, which is consumed in reaction 1 and produced in reaction 2. The two reactions can be coupled in the form of a third reaction, which we

can write as the sum of reactions 1 and 2, with the common intermediate Pi omitted from both sides of the equation:

Reaction 3: Glucose ATP 88n glucose 6-phosphate ADP Because more energy is released in reaction 2 than is consumed in reaction

1, reaction 3 is exergonic: some energy is released (G3 in Fig. 1–9b). Living cells thus make glucose 6-phosphate by catalyzing a direct reaction between glucose and ATP, in effect coupling reaction 1 to reaction 2. NH2 The coupling of exergonic reactions with endergonic ones is absolutely N C C N central to the energy exchanges in living systems. The mechanism by which O O O HC energy coupling occurs in biological reactions is via a shared intermediate. C CH We will see that reaction 2 in Figure 1–9b, the breakdown of adenosine OPOPOPOCH2 N N O triphosphate (ATP), is the exergonic reaction that drives many ender- O O O H H gonic processes in cells. In fact, ATP (Fig. 1–10) is the major carrier of H H chemical energy in all cells, coupling endergonic processes to exergonic OH OH ones. The terminal phosphoryl group of ATP, shaded pink in Figure 1–10, is transferred to a variety of acceptor molecules, which are thereby activated figure 1–10 for further chemical transformation. The adenosine diphosphate (ADP) Adenosine triphosphate (ATP). The removal of the terminal phosphoryl of ATP (shaded pink) is highly exergonic, that remains is recycled (phosphorylated) to ATP, at the expense of either and this reaction is coupled to many endergonic reactions chemical energy (during oxidation of fuels) or solar energy (in photosyn- in the cell as in the example described in Figure 1–9b. thetic cells). Chapter 1 The Molecular Logic of Life 11

Endergonic cellular reactions are driven by coupling them to exergonic chemical or photochemical processes through shared chemi- Activation barrier cal intermediates. (transition state, ‡) G

‡ Guncat Enzymes Promote Sequences of Chemical Reactions Reactants (A) ‡ Gcat An exergonic reaction does not necessarily proceed rapidly. The path from reactant(s) to product(s) almost invariably involves an energy barrier, Free energy, called the activation barrier (Fig. 1–11), that must be surmounted for any G Products (B) reaction to occur. The breaking of existing bonds and formation of new ones generally requires the distortion of the existing bonds, creating a transition state of higher free energy than either reactant or product. The high- Reaction coordinate (A B) est point in the reaction coordinate diagram represents the transition state. figure 1–11 Virtually every cellular chemical reaction occurs at a measurable rate Energy changes during a chemical reaction. An activa- only because of the presence of enzymes—biocatalysts that, like all other tion barrier, representing the transition state, must be catalysts, greatly enhance the rate of specific chemical reactions without overcome in the conversion of reactants (A) into products being consumed in the process. Enzymes lower the energy barrier between (B), even though the products are more stable than the reactant and product. The activation energy (G‡; Fig. 1–11) required to reactants, as indicated by a large, negative free-energy change (G). The energy required to overcome the acti- overcome this energy barrier could in principle be supplied by heating the vation barrier is the activation energy (G‡). Enzymes cat- reaction mixture, thereby increasing the kinetic energy of the molecules, alyze reactions by lowering the activation barrier. They the frequency with which they collide, and the likelihood that they will re- bind the transition-state intermediates tightly, and the act. However, this option is not available in living cells, which generally binding energy of this interaction effectively reduces the ‡ ‡ maintain a constant temperature. In fact, many cell components (proteins, activation energy from Guncat to G cat. (Note that the activation energy is unrelated to the free-energy change membranes) are inactivated by temperatures only a few degrees above an of the reaction, G.) organism’s normal internal temperature. Instead, enzymes speed reactions by taking advantage of binding effects. Two or more reactants bind to the enzyme’s surface close to each other and with stereospecific orientations that favor the reaction between them. Through this combination of prox- imity and orientation, the probability of productive collisions between reactants is increased by orders of magnitude relative to the uncatalyzed With enzyme process, when reactants are randomly oriented and distributed throughout an aqueous solution. Furthermore, the reactants themselves, in the process (B) of binding to the enzyme, undergo changes in shape that distort them to- Reaction: A B ward the transition state, thereby lowering the activation energy and enormously accelerating the rate of the reaction (Fig. 1–12). The relationship between the activation energy and reaction rate is exponential; a small de- crease in G‡ results in a very large increase in reaction rate. Enzyme- 10 14 Without enzyme (uncatalyzed) catalyzed reactions commonly proceed at rates up to 10 to 10 times Amount of product formed faster than uncatalyzed reactions. Metabolic catalysts are, with a few exceptions, proteins. (In a few cases, Time RNA molecules have catalytic roles, as discussed in Chapter 26.) Again with figure 1–12 a few exceptions, each enzyme protein catalyzes a specific reaction, and An enzyme increases the rate of a specific chemical each reaction in a cell is catalyzed by a different enzyme. Thousands of dif- reaction. In the presence of an enzyme specific for the ferent enzymes are therefore required by each cell. The multiplicity of en- conversion of reactant A into product B, the rate of the reaction may increase by many orders of magnitude zymes, their specificity (the ability to discriminate between reactants), and (powers of ten) over that of the uncatalyzed reaction. Like their susceptibility to regulation give cells the capacity to lower activation all catalysts, the enzyme is not consumed in the process; barriers selectively. This selectivity is crucial for the effective regulation of one enzyme molecule can act repeatedly to convert many cellular processes. molecules of A to B. 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. 1–13). Some pathways degrade organic nutrients figure 1–13 A linear metabolic pathway. In this pathway, the reactant A is converted in five steps into the product F, with each enzyme 1 enzyme 2 enzyme 3 enzyme 4 enzyme 5 A B C D E F step catalyzed by an enzyme specific for that reaction. 12 Part I Foundations of Biochemistry

figure 1–14 Stored Other nutrients cellular work ATP is the shared chemical intermediate linking energy- releasing to energy-requiring cell processes. Its role in Ingested Complex the cell is analogous to that of money in an economy: foods biomolecules it is “earned/produced” in exergonic reactions and “spent/consumed” in endergonic ones. Solar Mechanical photons work into simple end products in order to extract chemical energy and convert it Osmotic work into a form useful to the cell. Together these degradative, free-energy-yielding reactions are designated catabolism. Other pathways start with 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 overall network of enzyme-catalyzed pathways con- ADP stitutes cellular metabolism. ATP is the major connecting link (the shared 2 intermediate) between the catabolic and anabolic components of this net- Catabolic HPO4 Anabolic work (Fig. 1–14). The linked systems of enzyme-catalyzed reactions that reaction reaction pathways pathways act on the main constituents of cells—proteins, fats, sugars, and nucleic (exergonic) (endergonic) ATP acids—are virtually identical in all living organisms. ATP is the universal carrier of metabolic energy, linking catabolic and anabolic pathways.

Metabolism Is Regulated to Achieve Balance and Economy Not only do living cells simultaneously synthesize thousands of different kinds of carbohydrate, fat, protein, and nucleic acid molecules and their simpler subunits, they do so in the precise proportions required by the cell. CO2 For example, during rapid cell growth, the precursors of proteins and nu- NH3 cleic acids must be made in large quantities, whereas in nongrowing cells

S H2O rs the requirement for these precursors is much reduced. Key enzymes in im so ple cur each metabolic pathway are regulated so that each type of precursor mole- products, pre cule is produced in a quantity appropriate to the current requirements of the cell. Consider the pathway that leads to the synthesis of isoleucine, one of the amino acids, the monomeric subunits of proteins (Fig. 1–15). 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 slow- ing the production of the amino acid. Such feedback inhibition keeps the production and utilization of each metabolic intermediate in balance. figure 1–15 Feedback inhibition. Regulation by feedback inhibition in a typical synthetic (anabolic) pathway. In the bacterium enzyme 1 E. coli, the amino acid threonine is converted to another A BCDEF amino acid, isoleucine, in five steps, each catalyzed by a Threonine Isoleucine separate enzyme. (The letters A through F represent the compounds, or intermediates, in this pathway.) The accumulation of the product isoleucine (F) causes inhibition of Living cells also regulate the synthesis of their own catalysts, the en- the first reaction in the pathway by binding to the enzyme catalyzing this reaction and reducing its activity. zymes. Thus a cell can switch off the synthesis of an enzyme required to make a given product whenever that product is adequately supplied. 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, continually adjusting for maximum economy. Chapter 1 The Molecular Logic of Life 13

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, be 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, deoxyribonucleic acid, or 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 organ- figure 1–16 isms is their ability to reproduce themselves with nearly perfect ﬁdelity for Two ancient scripts. (a) The Prism of Sennacherib, countless generations. This continuity of inherited traits implies constancy, inscribed in about 700 B.C., describes in characters of over thousands or millions of years, in the structure of the molecules that the Assyrian language some historical events during the contain the genetic information. Very few historical records of civilization, reign of King Sennacherib. The Prism contains about even those etched in copper or carved in stone, have survived for a thou- 20,000 characters, weighs about 50 kg, and has survived almost intact for about 2,700 years. (b) The sand years. But there is good evidence that the genetic instructions in liv- single DNA molecule of the bacterium E. coli, seen ing organisms have remained nearly unchanged over very much longer pe- leaking out of a disrupted cell, is hundreds of times riods; many bacteria have nearly the same size, shape, and internal longer than the cell itself and contains all of the encoded structure and contain the same kinds of precursor molecules and enzymes information necessary to specify the cell’s structure and as those that lived a billion years ago (Fig. 1–16). functions. The bacterial DNA contains about 10 million characters (nucleotides), weighs less than 1010 g, and has undergone only relatively minor changes during the past several million years. The yellow spots and dark specks in this colorized electron micrograph are artifacts of the preparation.

(a) (b) 14 Part I Foundations of Biochemistry

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 dupli- cate it for the next generation results from the structural complementarity between the two halves of the DNA molecule (Fig. 1–17). The basic unit of DNA is a linear polymer of four different monomeric subunits, deoxyribonucleotides (Fig. 1–3), arranged in a precise linear sequence. It is this linear sequence that encodes the genetic information. Two of these poly- meric strands are twisted about each other to form the DNA double helix, in which each monomeric subunit in one strand pairs speciﬁcally with a complementary subunit in the opposite strand. Before a cell divides, the Strand 1 two DNA strands separate and each serves as a template for the synthesis of a new 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 in the other strand, Strand 2 which acts as a template for repair of the damage.

Genetic information is encoded in the linear sequence of four kinds of subunits of DNA. C G C G The double-helical DNA molecule contains an internal template T T for its own replication and repair.

C G C G T A T A G C G C Changes in the Hereditary Instructions Allow Evolution Despite the near-perfect ﬁdelity of genetic replication, infrequent, unre- paired mistakes in the replication process produce changes in the nu- T A T A cleotide sequence of DNA, representing a genetic mutation (Fig. 1–18). C G C G Incorrectly repaired damage to one of the DNA strands has the same effect. G C G C Mutations can change the instructions for producing cellular components. T T Many mutations are harmful or even lethal to the organism; they may, for example, cause the synthesis of a defective enzyme that is not able to cat- G C G C alyze an essential metabolic reaction. Occasionally, a mutation better equips T A T A an organism or cell to survive in its environment. The mutant enzyme G C G C Old New New Old strand 1 strand 2 strand 1 strand 2 figure 1–17 The complementary structure of DNA. Complementarity sequence in the other; wherever G occurs in strand 1, C between the two strands accounts for the accurate repli- occurs in strand 2; wherever A occurs in strand 1, T cation essential for genetic continuity. DNA is a linear occurs in strand 2. The two strands of the DNA, held polymer of covalently joined subunits, the four deoxyribo- together by a large number of hydrogen bonds (repre- nucleotides: deoxyadenylate (A), deoxyguanylate (G), sented here by vertical blue lines) between the pairs of deoxycytidylate (C), and deoxythymidylate (T). Each complementary nucleotides, twist about each other to nucleotide has the intrinsic ability, due to its precise form the DNA double helix. In DNA replication, prior to three-dimensional structure, to associate very speciﬁcally cell division, the two strands of the original DNA separate but noncovalently with one other nucleotide in the comple- and two new strands are synthesized, each with a mentary chain: A always associates with its complement sequence complementary to one of the original strands. T, and G with its complement C. Thus, in the double- The result is two double-helical DNA molecules, each stranded DNA molecule, the entire sequence of identical to the original DNA. nucleotides in one strand is complementary to the Chapter 1 The Molecular Logic of Life 15

figure 1–18 Time Role of mutation in evolution. The gradual accumulation of mutations over long periods of time results in new bio- T G A G C T A logical species, each with a unique DNA sequence. At the top is shown a short segment of a gene in a hypothetical Mutation progenitor organism. With the passage of time, changes 1 in nucleotide sequence (mutations, indicated here by colored boxes) occur, one nucleotide at a time, resulting T G A A C T A TGA G CTA in progeny with different DNA sequences. These mutant progeny themselves undergo occasional mutations, yielding Mutation their own progeny differing by two or more nucleotides 2 from the original sequence. When two lineages have changed so much by this mechanism that they can no T G A A C G A T GAA C T A longer interbreed, a new species has been created.

Mutation Mutation 3 4

Mutation T C A G C T A GGA G CTA 5

T T A A C G A T GAA C G A

Mutation 6

TACTC GTG C A G CTA

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 and reproduction 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 ecologi- cal niche. Carolus Linnaeus 1707–1778 Molecular Anatomy Reveals Evolutionary Relationships The eighteenth-century naturalist Carolus Linnaeus recognized the anatomic similarities and differences among living organisms and provided a framework for assessing the relatedness of species. Charles Darwin, in the nineteenth century, gave us a unifying hypothesis to explain the phylogeny of modern organisms—the origin of different species from a common an- cestor. Now biochemical research in the twentieth century has revealed the molecular anatomy of cells of different species—the subunit sequences and the three-dimensional structures of individual nucleic acids and proteins. Biochemists have an enormously rich and increasing treasury of evidence that can be used to analyze evolutionary relationships and to refine evolu- Charles Darwin tionary theory. The nucleotide sequences of entire genomes (the genome is 1809–1882 16 Part I Foundations of Biochemistry

table 1–1 Some Organisms Whose Genomes Have Been Completely Sequenced Genome size Organism (million bases) Biological interest Mycoplasma pneumoniae 0.8 Causes pneumonia Treponema pallidum 1.1 Causes syphilis Borrelia burgdorferi 1.3 Causes Lyme disease Helicobacter pylori 1.7 Causes gastric ulcers Methanococcus jannaschii 1.7 Grows at 85 C! Haemophilus inﬂuenzae 1.8 Causes bacterial inﬂuenza Methanobacterium thermo- 1.8 Member of the Archaea autotrophicum Archaeoglobus fulgidus 2.2 High-temperature methanogen Synechocystis sp. 3.6 Cyanobacterium Bacillus subtilis 4.2 Common soil bacterium Escherichia coli 4.6 Some strains cause toxic shock syndrome Saccharomyces cerevisiae 12.1 Unicellular eukaryote Caenorhabditis elegans 97.1 Multicellular roundworm

the complete genetic endowment of an organism) have been determined (Table 1–1). The genomic sequences of a number of eubacteria and an archaebacterium, a eukaryotic microorganism (Saccharomyces cerevisiae), and a multicellular animal (Caenorhabditis elegans) have been determined; those of a plant (Arabidopsis thaliana) and even of Homo sapiens will soon be known. With such sequences in hand, highly detailed and quantitative comparisons among species will provide deep insight into the evolutionary process. Thus far, 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 possible the discovery of the unifying features common to all life.

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 subunits, 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 an intermediary, RNA) for the production of a protein with a cor- responding linear sequence of amino acids (Fig. 1–19). The protein folds into a particular three-dimensional shape, determined by its amino acid sequence and stabilized primarily by noncovalent interactions. Although the ﬁnal shape of the folded protein is dictated by its amino acid sequence, the folding process is aided by proteins that act as “molecular chaperones,” dis- couraging incorrect folding. The precise three-dimensional structure, or native conformation, is crucial to the protein’s function. Chapter 1 The Molecular Logic of Life 17

figure 1–19 Gene 1 Gene 2 Gene 3 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 into their native three-dimensional shapes, often aided by other proteins called molecular chaperones. Individual proteins commonly associate with other proteins to form supramol- Transcription of DNA sequence ecular complexes, stabilized by numerous into RNA sequence weak interactions.

RNA 1 RNA 2 RNA 3 The linear sequence of amino acids in a protein leads to the acqui- sition of a unique three-dimensional structure.

Translation on the ribosome of RNA sequence Once a protein has folded into its native conformation, it may associ- into protein sequence and folding of protein ate noncovalently with other proteins, or with nucleic acids or lipids, to into native conformation form supramolecular complexes such as chromosomes, ribosomes, and membranes. 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.

Protein 1 Protein 2 Protein 3 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 collec- Formation of supramolecular complex tively 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 gives macromolecules a ﬂexibility 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.

For example, the double-helical DNA molecule, with its complementary strands held together by many weak interactions, has enough flexibility to allow strand separation during DNA replication (Fig. 1–17), yet enough stability to ensure 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 enzyme-substrate complex survives the structural distortions that occur as the reactant is converted into product. 18 Part I Foundations of Biochemistry

Noncovalent interactions provide the energy for self-assembly of macromolecules by stabilizing their native conformations relative to their unfolded, random forms. A protein will assume this more stable shape, its native conformation, when the energetic advantages of forming weak interactions outweigh the tendency of the protein chain to assume random forms.

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 constant-temperature system of molecules that ex- tracts free energy and raw materials from its environment. The cell uses this energy to maintain itself in a dynamic steady state, far from equilibrium with its surroundings. The many chemical transformations within cells are organized into a network of reaction pathways, promoted at each step by specific catalysts, called enzymes, which the cell itself produces. A great economy of parts and processes is achieved by regulation of the 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 biological macromolecules and supramolecular complexes, while allowing sufficient flexibility for biological actions.

The chemical reactions and regulatory processes of cells have been highly refined over the course of billions of years of evolution. Nevertheless, no matter how complex it may seem, the organic machinery of living cells functions within the same set of physical laws that governs the operation of inanimate machines. This set of principles has been most thoroughly validated in studies of unicellular organisms (such as the bacterium E. coli), which are excep- tionally amenable to biochemical and genetic investigation. Multicellular organisms must solve certain problems not encountered by unicellular organisms, such as the differentiation of the fertilized egg into specialized cell types. Yet here, too, 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 organisms 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 Chapter 1 The Molecular Logic of Life 19 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.