8885dc06190-2371/27/047:13 AM Page190 chapter ENZYMES 6.1 An Introduction to Enzymes 191 H2O in the presence of oxygen is a highly exergonic 6.2 How Enzymes Work 193 process, releasing free energy that we can use to think, move, taste, and see. However, a bag of sugar can re- 6.3 Enzyme Kinetics as an Approach to Understanding main on the shelf for years without any obvious con Mechanism 202 version to CO2 and H.O. Although this chemical process 6.4 Examples of Enzymatic Reactions 213 is thermodynamically favorable, it is very slow! Yet when 6.5 Regulatory Enzymes 225 sucrose is consumed by a human (or almost any other organism), it releases its chemical energy in seconds The difference is catalysis. Without catalysis, chemical reactions such as sucrose oxidation could not occur on One way in which this condition might be fulfilled would a useful time scale and thus could not sustain life be if the molecules when combined with the enzyme, lay In this chapter, then, we turn our attention to the slightly further apart than their equilibrium distance when reaction catalysts of biological systems: the enzymes, [covalently joined], but nearer than their equilibrium distance when free.... Using fischers lock and ke Enzymes have extraordinary catalytic power, often far greater than that of synthetic or inorganic catalysts simile, the key does not fit the lock quite perfectly but They have a high degree of specificity for their sub- exercises a certain strain on it strates, they accelerate chemical reactions tremen- . B S Haldane, Enzymes, 1930 dously, and they function in aqueous solutions under very mild conditions of temperature and pH Few non- Catalysis can be described formally in terms of a biological catalysts have all these properties stabilization of the transition state through tight binding to Enzymes are central to every biochemical process. Acting in organized sequences, they catalyze the the catalyst. hundreds of stepwise reactions that degrade nutrient William P Jencks, article in Advances in Enzymology, 1975 molecules, conserve and transform chemical energy, and make biological macromolecules from simple pre cursors. Through the action of regulatory enzymes metabolic pathways are highly coordinated to yield a here are two fundamental conditions for life. First, harmonious interplay among the many activities ne the living entity must be able to self-replicate(a top essary to sustain life onsidered in Part ID; second, the organism must be The study of enzymes has immense practical im- able to catalyze chemical reactions efficiently and se portance. In some diseases, especially inheritable ge- lectively. The central importance of catalysis may sur- netic disorders, there may be a deficiency or even a prise some beginning students of biochemistry, but it is total absence of one or more enzymes. For other dis easy to demonstrate. As described in Chapter 1, living ease conditions, excessive activity of an enzyme may be ystems make use of energy from the environment he cause. Measurements of the activities of enzymes in Many of us, for example, consume substantial amounts blood plasma, erythrocytes, or tissue samples are im- of sucrose---common table sugar-as a kind of fuel, portant in diagnosing certain illnesses. Many drugs ex- whether in the form of sweetened foods and drinks or ert their biological effects through interactions with as sugar itself. The conversion of sucrose to COe and enzymes. And enzymes are important practical tools
chapter T here are two fundamental conditions for life. First, the living entity must be able to self-replicate (a topic considered in Part III); second, the organism must be able to catalyze chemical reactions efficiently and selectively. The central importance of catalysis may surprise some beginning students of biochemistry, but it is easy to demonstrate. As described in Chapter 1, living systems make use of energy from the environment. Many of us, for example, consume substantial amounts of sucrose—common table sugar—as a kind of fuel, whether in the form of sweetened foods and drinks or as sugar itself. The conversion of sucrose to CO2 and H2O in the presence of oxygen is a highly exergonic process, releasing free energy that we can use to think, move, taste, and see. However, a bag of sugar can remain on the shelf for years without any obvious conversion to CO2 and H2O. Although this chemical process is thermodynamically favorable, it is very slow! Yet when sucrose is consumed by a human (or almost any other organism), it releases its chemical energy in seconds. The difference is catalysis. Without catalysis, chemical reactions such as sucrose oxidation could not occur on a useful time scale, and thus could not sustain life. In this chapter, then, we turn our attention to the reaction catalysts of biological systems: the enzymes, the most remarkable and highly specialized proteins. Enzymes have extraordinary catalytic power, often far greater than that of synthetic or inorganic catalysts. They have a high degree of specificity for their substrates, they accelerate chemical reactions tremendously, and they function in aqueous solutions under very mild conditions of temperature and pH. Few nonbiological catalysts have all these properties. Enzymes are central to every biochemical process. Acting in organized sequences, they catalyze the hundreds of stepwise reactions that degrade nutrient molecules, conserve and transform chemical energy, and make biological macromolecules from simple precursors. Through the action of regulatory enzymes, metabolic pathways are highly coordinated to yield a harmonious interplay among the many activities necessary to sustain life. The study of enzymes has immense practical importance. In some diseases, especially inheritable genetic disorders, there may be a deficiency or even a total absence of one or more enzymes. For other disease conditions, excessive activity of an enzyme may be the cause. Measurements of the activities of enzymes in blood plasma, erythrocytes, or tissue samples are important in diagnosing certain illnesses. Many drugs exert their biological effects through interactions with enzymes. And enzymes are important practical tools, ENZYMES 6.1 An Introduction to Enzymes 191 6.2 How Enzymes Work 193 6.3 Enzyme Kinetics as an Approach to Understanding Mechanism 202 6.4 Examples of Enzymatic Reactions 213 6.5 Regulatory Enzymes 225 One way in which this condition might be fulfilled would be if the molecules when combined with the enzyme, lay slightly further apart than their equilibrium distance when [covalently joined], but nearer than their equilibrium distance when free. . . . Using Fischer’s lock and key simile, the key does not fit the lock quite perfectly but exercises a certain strain on it. —J. B. S. Haldane, Enzymes, 1930 Catalysis can be described formally in terms of a stabilization of the transition state through tight binding to the catalyst. —William P. Jencks, article in Advances in Enzymology, 1975 6 190 8885d_c06_190-237 1/27/04 7:13 AM Page 190 mac76 mac76:385_reb:
8885dc06190-2371/27/047:13 AM Page191mac76mac76:385 6.1 An Introduction to Enzymes 191 not only in medicine but in the chemical industry, food structure and chemical mechanism of many of them, and processing, and agriculture a general understanding of how enzymes work. e begin with descriptions of the properties of en- zymes and the principles underlying their catalytic Most Enzymes Are Proteins power, then introduce enzyme kinetics, a discipline that provides much of the framework for any discussion of with the exception of a small group of catalytic rNa nzymes. Specific examples of enzyme mechanisms are molecules(Chapter 26), all enzymes are proteins. Their then provided, illustrating principles introduced earlier catalytic activity depends on the integrity of their na- tive protein conformation. If an enzyme is denatured ol in the chapter. We end with a discussion of how enzyme dissociated into its subunits, catalytic activity is usually activity is regulated lost. If an enzyme is broken down into its component amino acids, its catalytic activity is always destroyed 6.1 An Introduction to Enzymes Thus the primary, secondary, tertiary, and quaternary structures of protein enzymes are essential to their cat Much of the history of biochemistry is the history of en- alytic activity zyme research. Biological catalysis was first recognized Enzymes, like other proteins, have molecular and described in the late 1700s. in studies on the di- weights ranging from about 12,000 to more than I mil- gestion of meat by secretions of the stomach, and re- lion. Some enzymes require no chemical groups for search continued in the 1800s with examinations of the activity other than their amino acid residues. Others conversion of starch to sugar by saliva and various plant require an additional chemical component called a extracts In the 1850s. Louis pasteur concluded that fer- cofactor--either one or more inorganic ions, such as mentation of sugar into alcohol by yeast is catalyzed by Fe, Mg, Mn-t, or Zn (Table 6-1), or a complex ferments. He postulated that these ferments were in- organic or metalloorganic molecule called a coenzyme separable from the structure of living yeast cells; this (Table 6-2). Some enzymes require both a coenzyme view, called vitalism, prevailed for decades. Then in 1897 Eduard Buchner discovered that yeast extracts could ferment sugar to alcohol, proving that fermentation was TABLE 6-1 Some Inorganic Elements That promoted by molecules that continued to function when Serve as Cofactors for Enzymes removed from cells. Frederick W. Kuhne called these molecules enzymes. As vitalistic notions of life were Cytochrome oxidase disproved, the isolation of new enzymes and the inves Fe or Fe Cytochrome oxidase, catalase, peroxidase tigation of their properties advanced the science of Pyruvate kinase biochemistry. Hexokinase, glucose 6-phosphatase, The isolation and crystallization of urease by James Sumner in 1926 provided a breakthrough in early enzyme Arginase, ribonucleotide reductase studies. Sumner found that urease crystals consisted Dinitrogenase entirely of protein, and he postulated that all enzymes Urease are proteins. In the absence of other examples, this Se Glutathione peroxidase idea remained controversial for some time. Only in the Carbonic anhydrase, alcohol 1930s was Sumner's conclusion widely accepted, after dehydrogenase, carboxypeptidases John Northrop and Moses Kunitz crystallized pepsin, A and B trypsin, and other digestive enzymes and found them also to be proteins. During this period L.B. S. Haldane wrote a treatise entitled Enzymes. Although the molecular nature of enzymes was not yet fully appreciated Haldane made the remarkable suggestion that weak bonding interactions between an enzyme and its substrate might be used to catalyze a reaction. This insight lies at the heart of our current under- Since the latter part of the twentieth century, research on enzymes has been intensive. It has led to the purification of Eduard Buchner, James Sumner 1. B S. Haldane thousands of enzymes, elucidation of the 1860-1917 1887-1955 1892-1964
not only in medicine but in the chemical industry, food processing, and agriculture. We begin with descriptions of the properties of enzymes and the principles underlying their catalytic power, then introduce enzyme kinetics, a discipline that provides much of the framework for any discussion of enzymes. Specific examples of enzyme mechanisms are then provided, illustrating principles introduced earlier in the chapter. We end with a discussion of how enzyme activity is regulated. 6.1 An Introduction to Enzymes Much of the history of biochemistry is the history of enzyme research. Biological catalysis was first recognized and described in the late 1700s, in studies on the digestion of meat by secretions of the stomach, and research continued in the 1800s with examinations of the conversion of starch to sugar by saliva and various plant extracts. In the 1850s, Louis Pasteur concluded that fermentation of sugar into alcohol by yeast is catalyzed by “ferments.” He postulated that these ferments were inseparable from the structure of living yeast cells; this view, called vitalism, prevailed for decades. Then in 1897 Eduard Buchner discovered that yeast extracts could ferment sugar to alcohol, proving that fermentation was promoted by molecules that continued to function when removed from cells. Frederick W. Kühne called these molecules enzymes. As vitalistic notions of life were disproved, the isolation of new enzymes and the investigation of their properties advanced the science of biochemistry. The isolation and crystallization of urease by James Sumner in 1926 provided a breakthrough in early enzyme studies. Sumner found that urease crystals consisted entirely of protein, and he postulated that all enzymes are proteins. In the absence of other examples, this idea remained controversial for some time. Only in the 1930s was Sumner’s conclusion widely accepted, after John Northrop and Moses Kunitz crystallized pepsin, trypsin, and other digestive enzymes and found them also to be proteins. During this period, J. B. S. Haldane wrote a treatise entitled Enzymes. Although the molecular nature of enzymes was not yet fully appreciated, Haldane made the remarkable suggestion that weak bonding interactions between an enzyme and its substrate might be used to catalyze a reaction. This insight lies at the heart of our current understanding of enzymatic catalysis. Since the latter part of the twentieth century, research on enzymes has been intensive. It has led to the purification of thousands of enzymes, elucidation of the structure and chemical mechanism of many of them, and a general understanding of how enzymes work. Most Enzymes Are Proteins With the exception of a small group of catalytic RNA molecules (Chapter 26), all enzymes are proteins. Their catalytic activity depends on the integrity of their native protein conformation. If an enzyme is denatured or dissociated into its subunits, catalytic activity is usually lost. If an enzyme is broken down into its component amino acids, its catalytic activity is always destroyed. Thus the primary, secondary, tertiary, and quaternary structures of protein enzymes are essential to their catalytic activity. Enzymes, like other proteins, have molecular weights ranging from about 12,000 to more than 1 million. Some enzymes require no chemical groups for activity other than their amino acid residues. Others require an additional chemical component called a cofactor—either one or more inorganic ions, such as Fe2, Mg2, Mn2, or Zn2 (Table 6–1), or a complex organic or metalloorganic molecule called a coenzyme (Table 6–2). Some enzymes require both a coenzyme 6.1 An Introduction to Enzymes 191 Cu2 Cytochrome oxidase Fe2 or Fe3 Cytochrome oxidase, catalase, peroxidase K Pyruvate kinase Mg2 Hexokinase, glucose 6-phosphatase, pyruvate kinase Mn2 Arginase, ribonucleotide reductase Mo Dinitrogenase Ni2 Urease Se Glutathione peroxidase Zn2 Carbonic anhydrase, alcohol dehydrogenase, carboxypeptidases A and B TABLE 6–1 Some Inorganic Elements That Serve as Cofactors for Enzymes Eduard Buchner, 1860–1917 James Sumner, 1887–1955 J. B. S. Haldane, 1892–1964 8885d_c06_190-237 1/27/04 7:13 AM Page 191 mac76 mac76:385_reb:
8885dc06_190-2371/27/047:13 AM Page19 6mac76:385 Chapter 6 Enzymes TABLE 6-2 Some Coenzymes That Serve as Transient Carriers of Specific Atoms or Functional Grou zyme Examples of chemical groups transferred Dietary precursor in mammals Biocytin Biotin Coenzyme A Acyl groups Pantothenic acid and other compounds 5-Deoxyadenosylcobalamin H atoms and alkyl groups Vitamin B12 penzyme B, Flavin adenine dinucleotide Electrons Riboflavin(vitamin B2) Electrons and acyl groups Not required in diet Nicotinamide adenine dinucleotide Hydride ion(:H) Nicotinic acid(niacin Pyridoxal phosphate Amino groups ridoxine(vitamin B6) Tetrahydrofolate One-carbon groups Folate Thiamine pyrophosphate Aldehydes Thiamine(vitamin B1) Note The structures and modes of action of these coenzymes are described in Part IL and one or more metal ions for activity. A coenzyme or tion, before the specific reaction catalyzed was known metal ion that is very tightly or even covalently bound For example, an enzyme known to act in the digestion to the enzyme protein is called a prosthetic group. a of foods was named pepsin, from the greek pepsis, " di- complete, catalytically active enzyme together with its gestion, and lysozyme was named for its ability to lyse bound coenzyme and/or metal ions is called a holon- bacterial cell walls. Still others were named for their zyme. The protein part of such an enzyme is called the source: trypsin, named in part from the greek tryein, apoenzyme or apoprotein Coenzymes act as tran-"to wear down, was obtained by rubbing pancreatic sient carriers of specific functional groups. Most are de- tissue with glycerin. Sometimes the same enzyme has rived from vitamins, organic nutrients required in small two or more names, or two different enzymes have the amounts in the diet. We consider coenzymes in more same name. Because of such ambiguities, and the ever- detail as we encounter them in the metabolic pathways increasing number of newly discovered enzymes discussed in Part Il. Finally, some enzyme proteins are biochemists, by international agreement, have adopted modified covalently by phosphorylation, glycosylation, a system for naming and classifying enzymes. This sys and other processes. Many of these alterations are in- tem divides enzymes into six classes, each with sub volved in the regulation of enzyme activity. classes, based on the type of reaction catalyzed ( Table 6-3). Each enzyme is assigned a four-part classification Enzymes Are Classified by the Reactions number and a systematic name, which identifies the re- They Catalyze action it catalyzes. As an example, the formal system- atic name of the enzyme catalyzing the reaction Many enzymes have been named by adding the suffix ase"to the name of their substrate or to a word or ATP+ D-glucose→→ADP+ D-glucose6 phosphate phrase describing their activity. Thus urease catalyzes is ATP: glucose phosphotransferase, which indicates that hydrolysis of urea, and dna polymerase catalyzes the it catalyzes the transfer of a phosphoryl group from ATP polymerization of nucleotides to form DNA. Other en- to glucose. Its Enzyme Commission number (E.C. zymes were named by their discovers for a broad func- number) is 2.7.1.1. The first number (2) denotes the TABLE 6-3 International Classification of Enzymes Class type of reaction catalyzed Oxidoreductases Transfer of electrons(hydride ions or H atoms) 123456 Group transfer reactions Hydrolases Hydrolysis reactions(transfer of functional groups to wate Lyases Addition of groups to double bonds, or formation of double bonds by removal of groups Transfer of groups within molecules to yield isomeric forms Formation of C--C,C-S,C-0, and C-N bonds by condensation reactions coupled to ATP cleavag Note: Most enzymes catalyze the transfer of electrons, atoms, or functional groups. They are therefore classified, given code numbers, and assigned names according to the type of transfer reaction, the group dono, and the group acceptor
and one or more metal ions for activity. A coenzyme or metal ion that is very tightly or even covalently bound to the enzyme protein is called a prosthetic group. A complete, catalytically active enzyme together with its bound coenzyme and/or metal ions is called a holoenzyme. The protein part of such an enzyme is called the apoenzyme or apoprotein. Coenzymes act as transient carriers of specific functional groups. Most are derived from vitamins, organic nutrients required in small amounts in the diet. We consider coenzymes in more detail as we encounter them in the metabolic pathways discussed in Part II. Finally, some enzyme proteins are modified covalently by phosphorylation, glycosylation, and other processes. Many of these alterations are involved in the regulation of enzyme activity. Enzymes Are Classified by the Reactions They Catalyze Many enzymes have been named by adding the suffix “-ase” to the name of their substrate or to a word or phrase describing their activity. Thus urease catalyzes hydrolysis of urea, and DNA polymerase catalyzes the polymerization of nucleotides to form DNA. Other enzymes were named by their discovers for a broad function, before the specific reaction catalyzed was known. For example, an enzyme known to act in the digestion of foods was named pepsin, from the Greek pepsis, “digestion,” and lysozyme was named for its ability to lyse bacterial cell walls. Still others were named for their source: trypsin, named in part from the Greek tryein, “to wear down,” was obtained by rubbing pancreatic tissue with glycerin. Sometimes the same enzyme has two or more names, or two different enzymes have the same name. Because of such ambiguities, and the everincreasing number of newly discovered enzymes, biochemists, by international agreement, have adopted a system for naming and classifying enzymes. This system divides enzymes into six classes, each with subclasses, based on the type of reaction catalyzed (Table 6–3). Each enzyme is assigned a four-part classification number and a systematic name, which identifies the reaction it catalyzes. As an example, the formal systematic name of the enzyme catalyzing the reaction ATP D-glucose 88n ADP D-glucose 6-phosphate is ATP:glucose phosphotransferase, which indicates that it catalyzes the transfer of a phosphoryl group from ATP to glucose. Its Enzyme Commission number (E.C. number) is 2.7.1.1. The first number (2) denotes the 192 Chapter 6 Enzymes Coenzyme Examples of chemical groups transferred Dietary precursor in mammals Biocytin CO2 Biotin Coenzyme A Acyl groups Pantothenic acid and other compounds 5-Deoxyadenosylcobalamin H atoms and alkyl groups Vitamin B12 (coenzyme B12) Flavin adenine dinucleotide Electrons Riboflavin (vitamin B2) Lipoate Electrons and acyl groups Not required in diet Nicotinamide adenine dinucleotide Hydride ion (:H) Nicotinic acid (niacin) Pyridoxal phosphate Amino groups Pyridoxine (vitamin B6) Tetrahydrofolate One-carbon groups Folate Thiamine pyrophosphate Aldehydes Thiamine (vitamin B1) Note: The structures and modes of action of these coenzymes are described in Part II. TABLE 6–2 Some Coenzymes That Serve as Transient Carriers of Specific Atoms or Functional Groups No. Class Type of reaction catalyzed 1 Oxidoreductases Transfer of electrons (hydride ions or H atoms) 2 Transferases Group transfer reactions 3 Hydrolases Hydrolysis reactions (transfer of functional groups to water) 4 Lyases Addition of groups to double bonds, or formation of double bonds by removal of groups 5 Isomerases Transfer of groups within molecules to yield isomeric forms 6 Ligases Formation of COC, COS, COO, and CON bonds by condensation reactions coupled to ATP cleavage Note: Most enzymes catalyze the transfer of electrons, atoms, or functional groups. They are therefore classified, given code numbers, and assigned names according to the type of transfer reaction, the group donor, and the group acceptor. TABLE 6–3 International Classification of Enzymes 8885d_c06_190-237 1/27/04 7:13 AM Page 192 mac76 mac76:385_reb:
8885dc061932/2/042:50 PM Page193mac76mac76:385reb 6.2 How Enzymes Work class name(transferase); the second number (o), the subclass(phosphotransferase); the third number(1),a phosphotransferase with a hydroxyl group as acceptor; and the fourth number (1), D-glucose as the phosphoryl group acceptor. For many enzymes, a trivial name is more commonly used-in this case hexokinase. A com- plete list and description of the thousands of known en- zymes is maintained by the Nomenclature Committee of the International Union of Biochemistry and molecular iology(www.chem.qmul.ac.uk/iubmb/enzyme).Th chapter is devoted primarily to principles and proper ties common to all enzymes SUMMARY 6.1 An Introduction to Enzymes 豳 a Life depends on the existence of powerful and specific catalysts: the enzymes. Almost every biochemical reaction is catalyzed by an enzyme I With the exception of a few catalytic RNAs, all known enzymes are proteins. Many require The enzyme chymotrypsin, with bour 7GCH). Some key active-site amino aci und sul in red(PDB ID appear as a red nonprotein coenzymes or cofactors for their catalytic function. potch on the enzyme surface a Enzymes are classified according to the type of substrate complex, whose existence was first proposed reaction they catalyze. All enzymes have formal by Charles-Adolphe Wurtz in 1880, is central to the ac- E.C. numbers and names, and most have trivial tion of enzymes. It is also the starting point for mathe nanes matical treatments that define the kinetic behavior of enzyme-catalyzed reactions and for theoretical descrip- tions of enzyme mechanisms 6.2 How Enzymes Work The enzymatic catalysis of reactions is essential to liv- Enzymes Affect Reaction Rates, Not Equilibria ing systems Under biologically relevant conditions, un- A simple enzymatic reaction might be written catalyzed reactions tend to be slow--most biological molecules are quite stable in the neutral- pH, mild- E+s= ES= EP= E+P temperature, aqueous environment inside cells. Fur- where E, S, and P represent the enzyme, substrate, and thermore, many common reactions in biochemistry product; ES and EP are transient complexes of the en- entail chemical events that are unfavorable or unlikely zyme with the substrate and with the product in the cellular environment. such as the transien To understand catalysis, we must first appreciate formation of unstable charged intermediates or the col- the important distinction between reaction equilibria and lision of two or more molecules in the precise orienta- reaction rates. The function of a catalyst is to increase tion required for reaction. Reactions required to digest the rate of a reaction Catalysts do not affect reaction food, send nerve signals, or contract a muscle simply do equilibria. Any reaction, such as s= P can be de- not occur at a useful rate without catalysis scribed by a reaction coordinate diagram(Fig. 6-2),a An enzyme circumvents these problems by provid- picture of the energy changes during the reaction. As ing a specific environment within which a given reac- discussed in Chapter 1, energy in biological systems is tion can occur more rapidly. The distinguishing feature described in terms of free energy, G. In the coordinate of an enzyme-catalyzed reaction is that it takes place diagram, the free energy of the system is plotted against within the confines of a pocket on the enzyme called the progress of the reaction(the reaction coordinate) the active site(Fig. 6-1). The molecule that is bound The starting point for either the forward or the reverse in the active site and acted upon by the enzyme is called reaction is called the ground state, the contribution to the substrate. The surface of the active site is lined the free energy of the system by an average molecule with amino acid residues with substituent groups that ( S or P) under a given set of conditions. To describe the bind the substrate and catalyze its chemical transfor- free-energy changes for reactions, chemists define a mation. Often, the active site encloses a substrate, se- standard set of conditions(temperature 298 K; partial questering it completely from solution. The enzyme- pressure of each gas I atm, or 101.3 kPa; concentration
class name (transferase); the second number (7), the subclass (phosphotransferase); the third number (1), a phosphotransferase with a hydroxyl group as acceptor; and the fourth number (1), D-glucose as the phosphoryl group acceptor. For many enzymes, a trivial name is more commonly used—in this case hexokinase. A complete list and description of the thousands of known enzymes is maintained by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (www.chem.qmul.ac.uk/iubmb/enzyme). This chapter is devoted primarily to principles and properties common to all enzymes. SUMMARY 6.1 An Introduction to Enzymes ■ Life depends on the existence of powerful and specific catalysts: the enzymes. Almost every biochemical reaction is catalyzed by an enzyme. ■ With the exception of a few catalytic RNAs, all known enzymes are proteins. Many require nonprotein coenzymes or cofactors for their catalytic function. ■ Enzymes are classified according to the type of reaction they catalyze. All enzymes have formal E.C. numbers and names, and most have trivial names. 6.2 How Enzymes Work The enzymatic catalysis of reactions is essential to living systems. Under biologically relevant conditions, uncatalyzed reactions tend to be slow—most biological molecules are quite stable in the neutral-pH, mildtemperature, aqueous environment inside cells. Furthermore, many common reactions in biochemistry entail chemical events that are unfavorable or unlikely in the cellular environment, such as the transient formation of unstable charged intermediates or the collision of two or more molecules in the precise orientation required for reaction. Reactions required to digest food, send nerve signals, or contract a muscle simply do not occur at a useful rate without catalysis. An enzyme circumvents these problems by providing a specific environment within which a given reaction can occur more rapidly. The distinguishing feature of an enzyme-catalyzed reaction is that it takes place within the confines of a pocket on the enzyme called the active site (Fig. 6–1). The molecule that is bound in the active site and acted upon by the enzyme is called the substrate. The surface of the active site is lined with amino acid residues with substituent groups that bind the substrate and catalyze its chemical transformation. Often, the active site encloses a substrate, sequestering it completely from solution. The enzymesubstrate complex, whose existence was first proposed by Charles-Adolphe Wurtz in 1880, is central to the action of enzymes. It is also the starting point for mathematical treatments that define the kinetic behavior of enzyme-catalyzed reactions and for theoretical descriptions of enzyme mechanisms. Enzymes Affect Reaction Rates, Not Equilibria A simple enzymatic reaction might be written E S ES EP E P (6–1) where E, S, and P represent the enzyme, substrate, and product; ES and EP are transient complexes of the enzyme with the substrate and with the product. To understand catalysis, we must first appreciate the important distinction between reaction equilibria and reaction rates. The function of a catalyst is to increase the rate of a reaction. Catalysts do not affect reaction equilibria. Any reaction, such as S P, can be described by a reaction coordinate diagram (Fig. 6–2), a picture of the energy changes during the reaction. As discussed in Chapter 1, energy in biological systems is described in terms of free energy, G. In the coordinate diagram, the free energy of the system is plotted against the progress of the reaction (the reaction coordinate). The starting point for either the forward or the reverse reaction is called the ground state, the contribution to the free energy of the system by an average molecule (S or P) under a given set of conditions. To describe the free-energy changes for reactions, chemists define a standard set of conditions (temperature 298 K; partial pressure of each gas 1 atm, or 101.3 kPa; concentration yz yz yz yz 6.2 How Enzymes Work 193 FIGURE 6–1 Binding of a substrate to an enzyme at the active site. The enzyme chymotrypsin, with bound substrate in red (PDB ID 7GCH). Some key active-site amino acid residues appear as a red splotch on the enzyme surface. 8885d_c06_193 2/2/04 2:50 PM Page 193 mac76 mac76:385_reb:
8885dc06190-2371/27/047:13 AM Page194mac76mac76:385 Chapter 6 Enzymes Transition state(+ either substrate or product is equally likely. The differ ence between the energy levels of the ground state and the transition state is the activation energy, AG+. The rate of a reaction reflects this activation energy: a higher activation energy corresponds to a slower reaction. Re- △G"° action rates can be increased by raising the tempera gRound ture, thereby increasing the number of molecules with sufficient energy to overcome the energy barrier. Alter state natively, the activation energy can be lowered by adding Reaction coordinate a catalyst(Fig. 6-3) Catalysts enhance reaction rates by lowering activation energies. FIGURE 6-2 Reaction coordinate diagram for a chemical reaction Enzymes are no exception to the rule that catalysts The free energy of the system is plotted against the progress of the re. do not affect reaction equilibria. The bidirectional ar- action S-P. A diagram of this kind is a description of the energy rows in Equation 6-1 make this point: any enzyme that hanges during the reaction, and the horizontal axis(reaction coor- catalyzes the reaction s-P also catalyzes the reaction dinate)reflects the progressive chemical changes (e., bond breakage P-S. The role of enzymes is to accelerate the inter or formation) as S is converted to P The activation energies, AG, for conversion of S and P. The enzyme is not used up in the the s-P and P-Sreactions are indicated AC" is the overall stan- process, and the equilibrium point is unaffected. How dard free-energy change in the direction S-P. ever, the reaction reaches equilibrium much faster when the appropriate enzyme is present, because the rate of the reaction is increased of each solute 1 M) and express the free-energy change This general principle can be illustrated by consid- for this reacting system as AG, the standard free- ering the conversion of sucrose and oxygen to carbon energy change Because biochemical systems commonly involve ht concentrations far below 1 M. biochemists define a biochemical standard free-energy change C12H22O1+1202→12CO2+11H2O AG, the standard free-energy change at pH 7.0, we This conversion, which takes place through a series of employ this definition throughout the book. A more separate reactions, has a very large and negative AG complete definition of AG is given in Chapter 13 and at equilibrium the amount of sucrose present is neg The equilibrium between S and P reflects the dif- ligible. Yet sucrose is a stable compound, because the ference in the free energies of their ground states. In activation energy barrier that must be overcome before the example shown in Figure 6-2, the free energy of the sucrose reacts with oxygen is quite high. Sucrose can ground state of P is lower than that of S, So AG for the be stored in a container with oxygen almost indefinitely reaction is negative and the equilibrium favors P. The without reacting. In cells, however, sucrose is readily position and direction of equilibrium are not affected by broken down to co and Ho in a series of reactions any catalyst catalyzed by enzymes. These enzymes not only accel A favorable equilibrium does not mean that the s-P conversion will occur at a detectable rate. The rate of a reaction is dependent on an entirely different parameter. There is an energy barrier between S and P Transition state(+) the energy required for alignment of reacting groups formation of transient unstable charges, bond re arrangements, and other transformations required for the reaction to proceed in either direction. This is il lustrated by the energy "hill"in Figures 6-2 and 6-3. To undergo reaction, the molecules must overcome this barrier and therefore must be raised to a higher energy level. At the top of the energy hill is a point at which decay to the s or P state is equally probable (it is down- Reaction coordinate hill either way). This is called the transition state. The FIgure 6-3 Reaction coordinat transition state is not a chemical species with any sig catalyzed and uncatalyzed reactions. In the reaction S-P, the ES nificant stability and should not be confused with a re- and EP intermediates occupy minima in the energy progress curve of action intermediate(such as Es or EP). It is simply a the enzyme-catalyzed reaction. The terms AGuncat and ACtat corre- fleeting molecular moment in which events such as bond spond to the activation energy for the uncatalyzed reaction and the breakage, bond formation, and charge development overall activation energy for the catalyzed reaction, respectively.The have proceeded to the precise point at which decay to activation energy is lower when the enzyme catalyzes the reaction
of each solute 1 M) and express the free-energy change for this reacting system as G, the standard freeenergy change. Because biochemical systems commonly involve H concentrations far below 1 M, biochemists define a biochemical standard free-energy change, G, the standard free-energy change at pH 7.0; we employ this definition throughout the book. A more complete definition of G is given in Chapter 13. The equilibrium between S and P reflects the difference in the free energies of their ground states. In the example shown in Figure 6–2, the free energy of the ground state of P is lower than that of S, so G for the reaction is negative and the equilibrium favors P. The position and direction of equilibrium are not affected by any catalyst. A favorable equilibrium does not mean that the S n P conversion will occur at a detectable rate. The rate of a reaction is dependent on an entirely different parameter. There is an energy barrier between S and P: the energy required for alignment of reacting groups, formation of transient unstable charges, bond rearrangements, and other transformations required for the reaction to proceed in either direction. This is illustrated by the energy “hill” in Figures 6–2 and 6–3. To undergo reaction, the molecules must overcome this barrier and therefore must be raised to a higher energy level. At the top of the energy hill is a point at which decay to the S or P state is equally probable (it is downhill either way). This is called the transition state. The transition state is not a chemical species with any significant stability and should not be confused with a reaction intermediate (such as ES or EP). It is simply a fleeting molecular moment in which events such as bond breakage, bond formation, and charge development have proceeded to the precise point at which decay to either substrate or product is equally likely. The difference between the energy levels of the ground state and the transition state is the activation energy, G‡ . The rate of a reaction reflects this activation energy: a higher activation energy corresponds to a slower reaction. Reaction rates can be increased by raising the temperature, thereby increasing the number of molecules with sufficient energy to overcome the energy barrier. Alternatively, the activation energy can be lowered by adding a catalyst (Fig. 6–3). Catalysts enhance reaction rates by lowering activation energies. Enzymes are no exception to the rule that catalysts do not affect reaction equilibria. The bidirectional arrows in Equation 6–1 make this point: any enzyme that catalyzes the reaction S n P also catalyzes the reaction P n S. The role of enzymes is to accelerate the interconversion of S and P. The enzyme is not used up in the process, and the equilibrium point is unaffected. However, the reaction reaches equilibrium much faster when the appropriate enzyme is present, because the rate of the reaction is increased. This general principle can be illustrated by considering the conversion of sucrose and oxygen to carbon dioxide and water: C12H22O11 12O2 88n 12CO2 11H2O This conversion, which takes place through a series of separate reactions, has a very large and negative G, and at equilibrium the amount of sucrose present is negligible. Yet sucrose is a stable compound, because the activation energy barrier that must be overcome before sucrose reacts with oxygen is quite high. Sucrose can be stored in a container with oxygen almost indefinitely without reacting. In cells, however, sucrose is readily broken down to CO2 and H2O in a series of reactions catalyzed by enzymes. These enzymes not only accel- 194 Chapter 6 Enzymes Transition state (‡) Free energy, G Reaction coordinate S Ground state P Ground state G‡ S P G‡ P S G FIGURE 6–2 Reaction coordinate diagram for a chemical reaction. The free energy of the system is plotted against the progress of the reaction Sn P. A diagram of this kind is a description of the energy changes during the reaction, and the horizontal axis (reaction coordinate) reflects the progressive chemical changes (e.g., bond breakage or formation) as S is converted to P. The activation energies, G‡ , for the Sn P and Pn S reactions are indicated. G is the overall standard free-energy change in the direction Sn P. Transition state (‡) Reaction coordinate S P G‡ uncat G‡ cat ‡ ES EP Free energy, G FIGURE 6–3 Reaction coordinate diagram comparing enzymecatalyzed and uncatalyzed reactions. In the reaction Sn P, the ES and EP intermediates occupy minima in the energy progress curve of the enzyme-catalyzed reaction. The terms G‡ uncat and G‡ cat correspond to the activation energy for the uncatalyzed reaction and the overall activation energy for the catalyzed reaction, respectively. The activation energy is lower when the enzyme catalyzes the reaction. 8885d_c06_190-237 1/27/04 7:13 AM Page 194 mac76 mac76:385_reb: