c06Enzymes.

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6
Enzymes
6.1 An Introduction to Enzymes 189 Enzymes are central to every biochemical process.
Acting in organized sequences, they catalyze the hun-
6.2 How Enzymes Work 192 dreds of stepwise reactions that degrade nutrient mol-
6.3 Enzyme Kinetics as an Approach to Understanding ecules, conserve and transform chemical energy, and
Mechanism 200 make biological macromolecules from simple precursors.
The study of enzymes has immense practical impor-
6.4 Examples of Enzymatic Reactions 214 tance. In some diseases, especially inheritable genetic
6.5 Regulatory Enzymes 226 disorders, there may be a deficiency or even a total
absence of one or more enzymes. Other disease condi-
tions may be caused by excessive activity of an enzyme.

T
here are two fundamental conditions for life. First, Measurements of the activities of enzymes in blood
the organism must be able to self-replicate (a topic plasma, erythrocytes, or tissue samples are important in
considered in Part III); second, it must be able to diagnosing certain illnesses. Many drugs act through
catalyze chemical reactions efficiently and selectively. interactions with enzymes. Enzymes are also important
The central importance of catalysis may seem surpris- practical tools in chemical engineering, food technology,
ing, but it is easy to demonstrate. As described in Chap- and agriculture.
ter 1, living systems make use of energy from the envi- We begin with descriptions of the properties of
ronment. Many of us, for example, consume substantial enzymes and the principles underlying their catalytic
amounts of sucrose—common table sugar—as a kind of power, then introduce enzyme kinetics, a discipline that
fuel, usually in the form of sweetened foods and drinks. provides much of the framework for any discussion of
The conversion of sucrose to CO2 and H2O in the pres- enzymes. Specific examples of enzyme mechanisms are
ence of oxygen is a highly exergonic process, releasing then provided, illustrating principles introduced earlier
free energy that we can use to think, move, taste, and in the chapter. We end with a discussion of how enzyme
see. However, a bag of sugar can remain on the shelf for activity is regulated.
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
6.1 An Introduction to Enzymes
by a human (or almost any other organism), it releases Much of the history of biochemistry is the history of
its chemical energy in seconds. The difference is cataly- enzyme research. Biological catalysis was first recog-
sis. Without catalysis, chemical reactions such as sucrose nized and described in the late 1700s, in studies on the
oxidation could not occur on a useful time scale, and digestion of meat by secretions of the stomach. Research
thus could not sustain life. continued in the 1800s with examinations of the conver-
In this chapter, then, we turn our attention to the sion of starch to sugar by saliva and various plant extracts.
reaction catalysts of biological systems: enzymes, the In the 1850s, Louis Pasteur concluded that fermentation
most remarkable and highly specialized proteins. of sugar into alcohol by yeast is catalyzed by “ferments.”
Enzymes have extraordinary catalytic power, often far He postulated that these ferments were inseparable
greater than that of synthetic or inorganic catalysts. They from the structure of living yeast cells; this view, called
have a high degree of specificity for their substrates, vitalism, prevailed for decades. Then in 1897 Eduard
they accelerate chemical reactions tremendously, and Buchner discovered that yeast extracts could ferment
they function in aqueous solutions under very mild sugar to alcohol, proving that fermentation was promoted
conditions of temperature and pH. Few nonbiological by molecules that continued to function when removed
catalysts have all these properties. from cells. Buchner’s experiment at once marked the
189
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190 Enzymes

groups (Table 6–2). Most are derived

from vitamins, organic nutrients required
in small amounts in the diet. We consider
coenzymes in more detail as we encoun-
ter them in the metabolic pathways dis-
cussed in Part II. Some enzymes require
both a coenzyme 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, catalyti-
Eduard Buchner, 1860–1917 James Sumner, 1887–1955 J. B. S. Haldane, 1892–1964 cally active enzyme together with its
bound coenzyme and/or metal ions is
end of vitalistic notions and the dawn of the science of called a holoenzyme. The protein part of such an enzyme
biochemistry. Frederick W. Kühne later gave the name is called the apoenzyme or apoprotein. Finally, some
enzymes (from the Greek enzymos, “leavened”) to the enzyme proteins are modified covalently by phosphory-
molecules detected by Buchner. lation, glycosylation, and other processes. Many of these
The isolation and crystallization of urease by James alterations are involved in the regulation of enzyme
Sumner in 1926 was a breakthrough in early enzyme activity.
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 Enzymes Are Classified by the Reactions
remained controversial for some time. Only in the 1930s They Catalyze
was Sumner’s conclusion widely accepted, after John Many enzymes have been named by adding the suffix
Northrop and Moses Kunitz crystallized pepsin, trypsin, “-ase” to the name of their substrate or to a word or
and other digestive enzymes and found them also to be phrase describing their activity. Thus urease catalyzes
proteins. During this period, J. B. S. Haldane wrote a hydrolysis of urea, and DNA polymerase catalyzes the
treatise titled Enzymes. Although the molecular nature polymerization of nucleotides to form DNA. Other
of enzymes was not yet fully appreciated, Haldane made enzymes were named by their discoverers for a broad
the remarkable suggestion that weak bonding interac- function, before the specific reaction catalyzed was
tions between an enzyme and its substrate might be known. For example, an enzyme known to act in the
used to catalyze a reaction. This insight lies at the heart digestion of foods was named pepsin, from the Greek
of our current understanding of enzymatic catalysis. pepsis, “digestion,” and lysozyme was named for its ability
Since the latter part of the twentieth century, thou- to lyse (break down) bacterial cell walls. Still others
sands of enzymes have been purified, their structures were named for their source: trypsin, named in part
elucidated, and their mechanisms explained.

Most Enzymes Are Proteins

With the exception of a small group of catalytic RNA TABLE 6–1 Some Inorganic Ions That Serve as
molecules (Chapter 26), all enzymes are proteins. Their Cofactors for Enzymes
catalytic activity depends on the integrity of their native Ions Enzymes
protein conformation. If an enzyme is denatured or dis- 2⫹
sociated into its subunits, catalytic activity is usually Cu Cytochrome oxidase
lost. If an enzyme is broken down into its component 2⫹ 3⫹
Fe or Fe Cytochrome oxidase, catalase,
amino acids, its catalytic activity is always destroyed. peroxidase
Thus the primary, secondary, tertiary, and quaternary
K⫹ Pyruvate kinase
structures of protein enzymes are essential to their
2⫹
catalytic activity. Mg Hexokinase, glucose 6-phosphatase,
Enzymes, like other proteins, have molecular pyruvate kinase
weights ranging from about 12,000 to more than 1 mil- Mn2⫹ Arginase, ribonucleotide reductase
lion. Some enzymes require no chemical groups for
activity other than their amino acid residues. Others Mo Dinitrogenase
require an additional chemical component called a Ni2⫹ Urease
cofactor—either one or more inorganic ions, such as 2⫹
Zn Carbonic anhydrase, alcohol
Fe2⫹, Mg2⫹, Mn2⫹, or Zn2⫹ (Table 6–1), or a complex
dehydrogenase, carboxypeptidases
organic or metalloorganic molecule called a coenzyme.
A and B
Coenzymes act as transient carriers of specific functional
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6.1 An Introduction to Enzymes 191

TABLE 6–2 Some Coenzymes That Serve as Transient Carriers of Specific Atoms or Functional Groups
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.

from the Greek tryein, “to wear down,” was obtained by and the fourth number (1), D-glucose as the phosphoryl
rubbing pancreatic tissue with glycerin. Sometimes the group acceptor. For many enzymes, a trivial name is
same enzyme has two or more names, or two different more frequently used—in this case, hexokinase. A com-
enzymes have the same name. Because of such ambi- plete list and description of the thousands of known
guities, and the ever-increasing number of newly dis- enzymes is maintained by the Nomenclature Committee
covered enzymes, biochemists, by international agree- of the International Union of Biochemistry and Molecular
ment, have adopted a system for naming and classifying Biology (www.chem.qmul.ac.uk/iubmb/enzyme). This
enzymes. This system divides enzymes into six classes, chapter is devoted primarily to principles and properties
each with subclasses, based on the type of reaction common to all enzymes.
catalyzed (Table 6–3). Each enzyme is assigned a four-
part classification number and a systematic name, SUMMARY 6.1 An Introduction to Enzymes
which identifies the reaction it catalyzes. As an exam-  Life depends on powerful and specific catalysts:
ple, the formal systematic name of the enzyme catalyz- enzymes. Almost every biochemical reaction is
ing the reaction catalyzed by an enzyme.
ATP 1 D-glucose ¡ ADP 1 D-glucose 6-phosphate  With the exception of a few catalytic RNAs, all
is ATP : glucose phosphotransferase, which indicates known enzymes are proteins. Many require
that it catalyzes the transfer of a phosphoryl group from nonprotein coenzymes or cofactors for their
ATP to glucose. Its Enzyme Commission number (E.C. catalytic function.
number) is 2.7.1.1. The first number (2) denotes the  Enzymes are classified according to the type of
class name (transferase); the second number (7), the reaction they catalyze. All enzymes have formal
subclass (phosphotransferase); the third number (1), a E.C. numbers and names, and most have trivial
phosphotransferase with a hydroxyl group as acceptor; names.

TABLE 6–3 International Classification of Enzymes

Class no. Class name 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 Cleavage of C—C, C—O, C—N, or other bonds by elimination, leaving double
bonds or rings, or addition of groups to double bonds
5 Isomerases Transfer of groups within molecules to yield isomeric forms
6 Ligases Formation of C—C, C—S, C—O, and C—N bonds by condensation reactions
coupled to cleavage of ATP or similar cofactor
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192 Enzymes

6.2 How Enzymes Work where E, S, and P represent the enzyme, substrate, and
product; ES and EP are transient complexes of the
The enzymatic catalysis of reactions is essential to living enzyme with the substrate and with the product.
systems. Under biologically relevant conditions, uncata- To understand catalysis, we must first appreciate the
lyzed reactions tend to be slow—most biological mole- important distinction between reaction equilibria and
cules are quite stable in the neutral-pH, mild-temperature, reaction rates. The function of a catalyst is to increase
aqueous environment inside cells. Furthermore, many the rate of a reaction. Catalysts do not affect reaction
common chemical processes are unfavorable or unlikely equilibria. (Recall that a reaction is at equilibrium when
in the cellular environment, such as the transient forma- there is no net change in the concentrations of reactants
tion of unstable charged intermediates or the collision of or products.) Any reaction, such as S z y P, can be
two or more molecules in the precise orientation required described by a reaction coordinate diagram (Fig. 6–2), a
for reaction. Reactions required to digest food, send nerve picture of the energy changes during the reaction. As
signals, or contract a muscle simply do not occur at a use- discussed in Chapter 1, energy in biological systems is
ful rate without catalysis. described in terms of free energy, G. In the coordinate
An enzyme circumvents these problems by providing diagram, the free energy of the system is plotted against
a specific environment within which a given reaction can the progress of the reaction (the reaction coordinate).
occur more rapidly. The distinguishing feature of an The starting point for either the forward or the reverse
enzyme-catalyzed reaction is that it takes place within the reaction is called the ground state, the contribution
confines of a pocket on the enzyme called the active site to the free energy of the system by an average molecule
(Fig. 6–1). The molecule that is bound in the active site (S or P) under a given set of conditions.
and acted upon by the enzyme is called the substrate.
The surface of the active site is lined with amino acid KEY CONVENTION: To describe the free-energy changes for
residues with substituent groups that bind the substrate reactions, chemists define a standard set of conditions
and catalyze its chemical transformation. Often, the active (temperature 298 K; partial pressure of each gas 1 atm, or
site encloses a substrate, sequestering it completely from 101.3 kPa; concentration of each solute 1 M) and express
solution. The enzyme-substrate complex, whose exis- the free-energy change for a reacting system under these
tence was first proposed by Charles-Adolphe Wurtz in conditions as G, the standard free-energy change.
1880, is central to the action of enzymes. It is also the Because biochemical systems commonly involve H con-
starting point for mathematical treatments that define the centrations far below 1 M, biochemists define a biochem-
kinetic behavior of enzyme-catalyzed reactions and for ical standard free-energy change, G, the standard
theoretical descriptions of enzyme mechanisms. free-energy change at pH 7.0; we employ this definition
throughout the book. A more complete definition of G
Enzymes Affect Reaction Rates, Not Equilibria is given in Chapter 13. ■
A simple enzymatic reaction might be written
The equilibrium between S and P reflects the dif-
E 1 S Δ ES Δ EP Δ E 1 P (6–1) ference 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

Transition state (‡)

Key active site

G‡
Free energy, G

amino acid Substrate S P

‡
residues GP S

S G
Ground P
state Ground
state

Reaction coordinate

FIGURE 6–2 Reaction coordinate diagram. The free energy of the sys-
tem is plotted against the progress of the reaction S S P. A diagram of
this kind is a description of the energy changes during the reaction, and
FIGURE 6–1 Binding of a substrate to an enzyme at the active site. the horizontal axis (reaction coordinate) reflects the progressive chemi-
The enzyme chymotrypsin with bound substrate (PDB ID 7GCH). Some cal changes (e.g., bond breakage or formation) as S is converted to P. The
key active-site amino acid residues appear as a red splotch on the activation energies, G‡, for the S S P and P S S reactions are indicated.
enzyme surface. G is the overall standard free-energy change in the direction S S P.
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6.2 How Enzymes Work 193

the reaction is negative (the reaction is exergonic) and reaction P S S. The role of enzymes is to accelerate the
at equilibrium there is more P than S (the equilibrium interconversion of S and P. The enzyme is not used up
favors P). The position and direction of equilibrium are in the process, and the equilibrium point is unaffected.
not affected by any catalyst. However, the reaction reaches equilibrium much faster
A favorable equilibrium does not mean that the when the appropriate enzyme is present, because the
S S P conversion will occur at a detectable rate. The rate of the reaction is increased.
rate of a reaction is dependent on an entirely different This general principle is illustrated in the conver-
parameter. There is an energy barrier between S and P: sion of sucrose and oxygen to carbon dioxide and water:
the energy required for alignment of reacting groups, C12H22O11 1 12O2 Δ 12CO2 1 11H2O
formation of transient unstable charges, bond rear-
rangements, and other transformations required for the This conversion, which takes place through a series of
reaction to proceed in either direction. This is illustrated separate reactions, has a very large and negative G,
by the energy “hill” in Figures 6–2 and 6–3. To undergo and at equilibrium the amount of sucrose present is
reaction, the molecules must overcome this barrier and negligible. Yet sucrose is a stable compound, because the
therefore must be raised to a higher energy level. At the activation energy barrier that must be overcome before
top of the energy hill is a point at which decay to the S sucrose reacts with oxygen is quite high. Sucrose can
or P state is equally probable (it is downhill either way). be stored in a container with oxygen almost indefinitely
This is called the transition state. The transition state without reacting. In cells, however, sucrose is readily
is not a chemical species with any significant stability broken down to CO2 and H2O in a series of reactions
and should not be confused with a reaction intermedi- catalyzed by enzymes. These enzymes not only accelerate
ate (such as ES or EP). It is simply a fleeting molecular the reactions, they organize and control them so that
moment in which events such as bond breakage, bond much of the energy released is recovered in other
formation, and charge development have proceeded to chemical forms and made available to the cell for other
the precise point at which decay to either substrate or tasks. The reaction pathway by which sucrose (and
product is equally likely. The difference between the other sugars) is broken down is the primary energy-
energy levels of the ground state and the transition yielding pathway for cells, and the enzymes of this
state is the activation energy, ⌬G‡. The rate of a reac- pathway allow the reaction sequence to proceed on a
tion reflects this activation energy: a higher activation biologically useful time scale.
energy corresponds to a slower reaction. Reaction rates Any reaction may have several steps, involving the
can be increased by raising the temperature and/or formation and decay of transient chemical species
pressure, thereby increasing the number of molecules called reaction intermediates.* A reaction intermedi-
with sufficient energy to overcome the energy barrier. ate is any species on the reaction pathway that has a
Alternatively, the activation energy can be lowered by finite chemical lifetime (longer than a molecular vibra-
adding a catalyst (Fig. 6–3). Catalysts enhance reac- tion, ,1013 second). When the S z y P reaction is cata-
tion rates by lowering activation energies. lyzed by an enzyme, the ES and EP complexes can be
Enzymes are no exception to the rule that catalysts considered intermediates, even though S and P are
do not affect reaction equilibria. The bidirectional stable chemical species (Eqn 6–1); the ES and EP com-
arrows in Equation 6–1 make this point: any enzyme plexes occupy valleys in the reaction coordinate diagram
that catalyzes the reaction S S P also catalyzes the (Fig. 6–3). Additional, less stable chemical intermedi-
ates often exist in the course of an enzyme-catalyzed
reaction. The interconversion of two sequential reaction
Transition state (‡)
intermediates thus constitutes a reaction step. When
‡
several steps occur in a reaction, the overall rate is
G uncat determined by the step (or steps) with the highest acti-
Free energy, G

‡
vation energy; this is called the rate-limiting step. In a
G‡cat
ES EP
simple case, the rate-limiting step is the highest-energy
S point in the diagram for interconversion of S and P. In
P practice, the rate-limiting step can vary with reaction
conditions, and for many enzymes several steps may
have similar activation energies, which means they are
Reaction coordinate all partially rate-limiting.
FIGURE 6–3 Reaction coordinate diagram comparing enzyme-catalyzed
and uncatalyzed reactions. In the reaction S S P, the ES and EP inter- *In this chapter, step and intermediate refer to chemical species in
mediates occupy minima in the energy progress curve of the enzyme- the reaction pathway of a single enzyme-catalyzed reaction. In the
context of metabolic pathways involving many enzymes (discussed in
catalyzed reaction. The terms G‡uncat and G‡cat correspond to the acti-
Part II), these terms are used somewhat differently. An entire enzy-
vation energy for the uncatalyzed reaction and the overall activation matic reaction is often referred to as a “step” in a pathway, and the
energy for the catalyzed reaction, respectively. The activation energy is product of one enzymatic reaction (which is the substrate for the next
lower when the enzyme catalyzes the reaction. enzyme in the pathway) is referred to as a pathway “intermediate.”
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194 Enzymes

Activation energies are energy barriers to chemical (one in which there is much more product than substrate
reactions. These barriers are crucial to life itself. The at equilibrium)—but as already noted, this does not
rate at which a molecule undergoes a particular reaction mean the reaction will proceed at a rapid rate.
decreases as the activation barrier for that reaction The rate of any reaction is determined by the con-
increases. Without such energy barriers, complex mac- centration of the reactant (or reactants) and by a rate
romolecules would revert spontaneously to much sim- constant, usually denoted by k. For the unimolecular
pler molecular forms, and the complex and highly reaction S S P, the rate (or velocity) of the reaction,
ordered structures and metabolic processes of cells V—representing the amount of S that reacts per unit
could not exist. Over the course of evolution, enzymes time—is expressed by a rate equation:
have developed to lower activation energies selectively V 5 k[S] (6–4)
for reactions that are needed for cell survival.
In this reaction, the rate depends only on the concentra-
Reaction Rates and Equilibria Have Precise tion of S. This is called a first-order reaction. The factor
k is a proportionality constant that reflects the probabil-
Thermodynamic Definitions ity of reaction under a given set of conditions (pH, tem-
Reaction equilibria are inextricably linked to the perature, and so forth). Here, k is a first-order rate
standard free-energy change for the reaction, G, constant and has units of reciprocal time, such as s1. If
and reaction rates are linked to the activation energy, a first-order reaction has a rate constant k of 0.03 s1,
G‡. A basic introduction to these thermodynamic rela- this may be interpreted (qualitatively) to mean that 3%
tionships is the next step in understanding how enzymes of the available S will be converted to P in 1 s. A reaction
work. with a rate constant of 2,000 s1 will be over in a small
An equilibrium such as S z y P is described by fraction of a second. If a reaction rate depends on the
an equilibrium constant, Keq, or simply K (p. 25). concentration of two different compounds, or if the
Under the standard conditions used to compare bio- reaction is between two molecules of the same com-
chemical processes, an equilibrium constant is denoted pound, the reaction is second order and k is a second-
Keq (or K): order rate constant, with units of M1s1. The rate equa-
[P] tion then becomes
K¿eq 5 (6–2)
[S] V 5 k[S1][S2] (6–5)
From thermodynamics, the relationship between Keq
and G can be described by the expression From transition-state theory we can derive an expres-
sion that relates the magnitude of a rate constant to the
¢G¿8 5 2RT ln K¿eq (6–3) activation energy:
where R is the gas constant, 8.315 J/mol ? K, and T is the kT 2¢G‡/RT
absolute temperature, 298 K (25 8C). Equation 6–3 is k5 e (6–6)
h
developed and discussed in more detail in Chapter 13. where k is the Boltzmann constant and h is Planck’s
The important point here is that the equilibrium constant constant. The important point here is that the relation-
is directly related to the overall standard free-energy ship between the rate constant k and the activation
change for the reaction (Table 6–4). A large negative energy ¢G‡ is inverse and exponential. In simplified
value for G98 reflects a favorable reaction equilibrium terms, this is the basis for the statement that a lower
activation energy means a faster reaction rate.
Now we turn from what enzymes do to how they
TABLE 6–4 Relationship between K9eq and DG98 do it.

Keq DG98 (kJ/mol)

6 A Few Principles Explain the Catalytic Power and
10 34.2
Specificity of Enzymes
105 28.5
Enzymes are extraordinary catalysts. The rate enhance-
104 22.8 ments they bring about are in the range of 5 to 17 orders
103 17.1 of magnitude (Table 6–5). Enzymes are also very spe-
102 11.4 cific, readily discriminating between substrates with
101 5.7 quite similar structures. How can these enormous and
highly selective rate enhancements be explained? What
1 0.0 is the source of the energy for the dramatic lowering of
101 5.7 the activation energies for specific reactions?
102 11.4 The answer to these questions has two distinct but
103 17.1 interwoven parts. The first lies in the rearrangement of
covalent bonds during an enzyme-catalyzed reaction.
Note: The relationship is calculated from DG98  RT ln Keq (Eqn 6–3). Chemical reactions of many types take place between
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6.2 How Enzymes Work 195

2. Weak interactions are optimized in the reaction

TABLE 6–5 Some Rate Enhancements Produced transition state; enzyme active sites are
by Enzymes complementary not to the substrates per se but to
Cyclophilin 105 the transition states through which substrates pass
as they are converted to products during an
Carbonic anhydrase 107
enzymatic reaction.
Triose phosphate isomerase 109
These themes are critical to an understanding of
Carboxypeptidase A 1011
enzymes, and they now become our primary focus.
Phosphoglucomutase 1012
Succinyl-CoA transferase 1013 Weak Interactions between Enzyme and Substrate Are
Urease 1014 Optimized in the Transition State
Orotidine monophosphate decarboxylase 1017 How does an enzyme use binding energy to lower the
activation energy for a reaction? Formation of the ES
substrates and enzymes’ functional groups (specific complex is not the explanation in itself, although some
amino acid side chains, metal ions, and coenzymes). of the earliest considerations of enzyme mechanisms
Catalytic functional groups on an enzyme may form a began with this idea. Studies on enzyme specificity car-
transient covalent bond with a substrate and activate it ried out by Emil Fischer led him to propose, in 1894,
for reaction, or a group may be transiently transferred that enzymes were structurally complementary to their
from the substrate to the enzyme. In many cases, these substrates, so that they fit together like a lock and key
reactions occur only in the enzyme active site. Covalent (Fig. 6–4). This elegant idea, that a specific (exclusive)
interactions between enzymes and substrates lower the interaction between two biological molecules is mediated
activation energy (and thereby accelerate the reaction)
by providing an alternative, lower-energy reaction path.
The specific types of rearrangements that occur are NADP⫹
Unbound
described in Section 6.4.
The second part of the explanation lies in the non-
covalent interactions between enzyme and substrate.
Recall from Chapter 4 that weak, noncovalent interac-
tions help stabilize protein structure and protein-protein
interactions. These same interactions are critical to the
formation of complexes between proteins and small mol-
ecules, including enzyme substrates. Much of the energy Tetrahydrofolate
required to lower activation energies is derived from
weak, noncovalent interactions between substrate and
enzyme. What really sets enzymes apart from most other
catalysts is the formation of a specific ES complex. The
interaction between substrate and enzyme in this com-
Bound
plex is mediated by the same forces that stabilize protein
structure, including hydrogen bonds and hydrophobic
and ionic interactions (Chapter 4). Formation of each
weak interaction in the ES complex is accompanied by
release of a small amount of free energy that stabilizes
the interaction. The energy derived from enzyme-sub-
strate interaction is called binding energy, ⌬GB. Its
significance extends beyond a simple stabilization of the
enzyme-substrate interaction. Binding energy is a
major source of free energy used by enzymes to lower
the activation energies of reactions.
Two fundamental and interrelated principles pro-
vide a general explanation for how enzymes use nonco- FIGURE 6–4 Complementary shapes of a substrate and its binding site
valent binding energy: on an enzyme. The enzyme dihydrofolate reductase with its substrate
NADP, unbound and bound; another bound substrate, tetrahydrofolate, is
1. Much of the catalytic power of enzymes is also visible (PDB ID 1RA2). In this model, the NADP binds to a pocket
ultimately derived from the free energy released in that is complementary to it in shape and ionic properties, an illustration of
forming many weak bonds and interactions between Emil Fischer's “lock and key” hypothesis of enzyme action. In reality, the
an enzyme and its substrate. This binding energy complementarity between protein and ligand (in this case, substrate) is
contributes to specificity as well as to catalysis. rarely perfect, as we saw in Chapter 5.
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196 Enzymes

by molecular surfaces with complementary shapes, has the substrate (Fig. 6–5b). The active site of this stickase
greatly influenced the development of biochemistry, is a pocket lined with magnets. To react (break), the
and such interactions lie at the heart of many biochem- stick must reach the transition state of the reaction, but
ical processes. However, the “lock and key” hypothesis the stick fits so tightly in the active site that it cannot
can be misleading when applied to enzymatic catalysis. bend, because bending would eliminate some of the
An enzyme completely complementary to its substrate magnetic interactions between stick and enzyme. Such
would be a very poor enzyme, as we can demonstrate. an enzyme impedes the reaction, stabilizing the sub-
Consider an imaginary reaction, the breaking of a strate instead. In a reaction coordinate diagram (Fig.
magnetized metal stick. The uncatalyzed reaction is 6–5b), this kind of ES complex would correspond to an
shown in Figure 6–5a. Let’s examine two imaginary energy trough from which the substrate would have dif-
enzymes—two “stickases”—that could catalyze this ficulty escaping. Such an enzyme would be useless.
reaction, both of which employ magnetic forces as a The modern notion of enzymatic catalysis, first
paradigm for the binding energy used by real enzymes. proposed by Michael Polanyi (1921) and Haldane
We first design an enzyme perfectly complementary to (1930), was elaborated by Linus Pauling in 1946 and by

(a) No enzyme
‡
Substrate Transition state Products

Free energy, G
(metal stick) (bent stick) (broken stick) ΔG‡uncat

S P
‡
S
P
Reaction coordinate

(b) Enzyme complementary to substrate

‡
Free energy, G

Enzyme ΔG‡uncat

Magnets ΔG‡cat
Few
products S
ES P ΔGM

ES
Reaction coordinate

(c) Enzyme complementary to transition state

‡
E
ΔGM
Free energy, G

ΔG‡uncat
‡

‡ ΔG‡
ES ⫹
S ES
P P
Reaction coordinate

FIGURE 6–5 An imaginary enzyme (stickase) designed to catalyze increase in free energy required to bend the stick. Reaction coordinate
breakage of a metal stick. (a) Before the stick is broken, it must first be diagrams (right) show the energy consequences of complementarity to
bent (the transition state). In both stickase examples, magnetic interac- substrate versus complementarity to transition state (EP complexes are
tions take the place of weak bonding interactions between enzyme and omitted). ⌬GM, the difference between the transition-state energies of
substrate. (b) A stickase with a magnet-lined pocket complementary in the uncatalyzed and catalyzed reactions, is contributed by the magnetic
structure to the stick (the substrate) stabilizes the substrate. Bending is interactions between the stick and stickase. When the enzyme is com-
impeded by the magnetic attraction between stick and stickase. (c) An plementary to the substrate (b), the ES complex is more stable and has
enzyme with a pocket complementary to the reaction transition state less free energy in the ground state than substrate alone. The result is an
helps to destabilize the stick, contributing to catalysis of the reaction. increase in the activation energy.
The binding energy of the magnetic interactions compensates for the
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6.2 How Enzymes Work 197

William P. Jencks in the 1970s: in order to catalyze reac- catalysis. The groups on the substrate that are involved
tions, an enzyme must be complementary to the reac- in these weak interactions can be at some distance from
tion transition state. This means that optimal interac- the bonds that are broken or changed. The weak inter-
tions between substrate and enzyme occur only in the actions formed only in the transition state are those that
transition state. Figure 6–5c demonstrates how such an make the primary contribution to catalysis.
enzyme can work. The metal stick binds to the stickase, The requirement for multiple weak interactions to
but only a subset of the possible magnetic interactions drive catalysis is one reason why enzymes (and some
are used in forming the ES complex. The bound sub- coenzymes) are so large. An enzyme must provide func-
strate must still undergo the increase in free energy tional groups for ionic, hydrogen-bond, and other inter-
needed to reach the transition state. Now, however, the actions, and also must precisely position these groups
increase in free energy required to draw the stick into a so that binding energy is optimized in the transition
bent and partially broken conformation is offset, or state. Adequate binding is accomplished most readily by
“paid for,” by the magnetic interactions (binding energy) positioning a substrate in a cavity (the active site)
that form between the enzyme and substrate in the where it is effectively removed from water. The size of
transition state. Many of these interactions involve parts proteins reflects the need for superstructure to keep
of the stick that are distant from the point of breakage; interacting groups properly positioned and to keep the
thus interactions between the stickase and nonreacting cavity from collapsing.
parts of the stick provide some of the energy needed to
catalyze stick breakage. This “energy payment” trans-
lates into a lower net activation energy and a faster
Binding Energy Contributes to Reaction
reaction rate. Specificity and Catalysis
Real enzymes work on an analogous principle. Some Can we demonstrate quantitatively that binding energy
weak interactions are formed in the ES complex, but accounts for the huge rate accelerations brought about
the full complement of such interactions between sub- by enzymes? Yes. As a point of reference, Equation 6–6
strate and enzyme is formed only when the substrate allows us to calculate that G‡ must be lowered by
reaches the transition state. The free energy (binding about 5.7 kJ/mol to accelerate a first-order reaction by
energy) released by the formation of these interactions a factor of 10, under conditions commonly found in
partially offsets the energy required to reach the top of cells. The energy available from formation of a single
the energy hill. The summation of the unfavorable weak interaction is generally estimated to be 4 to
(positive) activation energy G‡ and the favorable 30 kJ/mol. The overall energy available from a number
(negative) binding energy GB results in a lower net of such interactions is therefore sufficient to lower acti-
activation energy (Fig. 6–6). Even on the enzyme, the vation energies by the 60 to 100 kJ/mol required to
transition state is not a stable species but a brief point explain the large rate enhancements observed for many
in time that the substrate spends atop an energy hill. enzymes.
The enzyme-catalyzed reaction is much faster than the The same binding energy that provides energy for
uncatalyzed process, however, because the hill is much catalysis also gives an enzyme its specificity, the ability
smaller. The important principle is that weak binding to discriminate between a substrate and a competing
interactions between the enzyme and the substrate molecule. Conceptually, specificity is easy to distinguish
provide a substantial driving force for enzymatic from catalysis, but this distinction is much more diffi-
cult to make experimentally, because catalysis and
specificity arise from the same phenomenon. If an
‡ enzyme active site has functional groups arranged opti-
mally to form a variety of weak interactions with a par-
‡ ⌬GB ticular substrate in the transition state, the enzyme will
⌬Guncat
Free energy, G

‡ not be able to interact to the same degree with any

⌬G‡cat
other molecule. For example, if the substrate has a
ES EP hydroxyl group that forms a hydrogen bond with a spe-
S
cific Glu residue on the enzyme, any molecule lacking a
P
hydroxyl group at that particular position will be a
poorer substrate for the enzyme. In addition, any mol-
ecule with an extra functional group for which the
Reaction coordinate
enzyme has no pocket or binding site is likely to be
FIGURE 6–6 Role of binding energy in catalysis. To lower the activation excluded from the enzyme. In general, specificity is
energy for a reaction, the system must acquire an amount of energy derived from the formation of many weak interactions
equivalent to the amount by which G‡ is lowered. Much of this energy between the enzyme and its specific substrate molecule.
comes from binding energy (GB) contributed by formation of weak The importance of binding energy to catalysis can be
noncovalent interactions between substrate and enzyme in the transi- readily demonstrated. For example, the glycolytic enzyme
tion state. The role of GB is analogous to that of GM in Figure 6–5. triose phosphate isomerase catalyzes the interconversion