Examples of Enzymatic Reactions

This chapter has focused on the general principles of catalysis and an introduction to some of the kinetic parameters used to describe enzyme action. Principles and kinetics are combined in Box 8-3, which describes some of the evidence that reinforces the notion that binding energy and transition-state complementarity are central to enzymatic catalysis. We now turn to several examples of specific enzyme reaction mechanisms.
An understanding of the complete mechanism of action of a purified enzyme requires a knowledge of (1) the temporal sequence in which enzyme-bound reaction intermediates occur, (2) the structure of each intermediate and transition state, (3) the rates of interconversion between intermediates, (4) the structural relationship of the enzyme with each intermediate, and (5) the energetic contributions of all reacting and interacting groups with respect to intermediate complexes and transition states. There is probably no enzyme for which current understanding meets this standard exactly. Many decades of research, however, have produced mechanistic information about hundreds of enzymes, and in some cases this information is highly detailed.

Reaction Mechanisms Illustrate Principles

Mechanisms are presented for three enzymes: chymotrypsin, hexokinase, and tyrosyl-tRNA synthetase. These are chosen not necessarily because they are the best-understood enzymes or cover all possible classes of enzyme chemistry, but because they help to illustrate some general principles outlined in this chapter. The discussion concentrates on selected principles, along with some key experiments that have helped to bring them into focus. Much mechanistic detail and experimental evidence is omitted, and in no instance do the mechanisms described below provide a complete explanation for the catalytic rate enhancements brought about by these enzymes.
This enzyme is a protease (Mr 25,000) specific for peptide bonds adjacent to aromatic amino acid residues (see Table 6-7). The three-dimensional structure of chymotrypsin is shown in Figure 8-18, with functional groups in the active site emphasized. This enzyme reaction illustrates the principle of transition-state stabilization by an enzyme, and also provides a classic example of the use of general acid-base catalysis and covalent catalysis (Fig. 8-19, p. 226). An excellent example is seen in a series of substrates for the enzyme chymotrypsin (Fig. 1). Chymotrypsin normally catalyzes the hydrolysis of peptide bonds next to aromatic amino acids, and the substrates shown in Figure 1 are convenient smaller models for the natural substrates (long polypeptides and proteins; see Chapter 6). The additional chemical groups added in going from A to B to C are shaded in red. Note that the interaction between the enzyme and these added functional groups has a minimal effect on Km (which is taken here as a reflection of KS), but a large, positive effect on kcat and kcat/Km. This is what we would expect if the interaction occurred only in the transition state. Chymotrypsin is described in more detail beginning on page 223.
A complementary experimental approach to this problem is to modify the enzyme, eliminating certain enzyme-substrate interactions, by replacing specific amino acids through site-directed mutagenesis (Chapters 7 and 28). A good example is found in tyrosyl-tRNA synthetase (p. 227).

Transition-State Analogs

Even though transition states cannot be observed directly, chemists can often predict the approximate structure of a transition state based on accumulated knowledge about reaction mechanisms. The transition state by definition is transient and so unstable that direct measurement of the binding interaction between this species and the enzyme is impossible. In some cases, however, stable molecules can be designed that resemble transition states. These are called transition-state analogs. In principle, they should bind to an enzyme more tightly than the substrate binds in the ES complex, because they should fit in the active site better (i.e., form more weak interactions) than the substrate itself. The idea of transition-state analogs was suggested by Pauling in the 1940s, and it has been used for a number of enzymes. These experiments have the limitation that a transitionstate analog can never mimic a transition state perfectly. Analogs have been found, however, that bind an enzyme 102 to 106 times more tightly than the normal substrate, providing good evidence that enzyme active sites are indeed complementary to transition states.

Catalytic Antibodies

If a transition-state analog can be designed for the reaction S → P, then an antibody that binds tightly to the transition-state analog might catalyze S → P. Antibodies (immunoglobulins; see Fig. 6-8) are key components of the immune response. A molecule or chemical group that is bound tightly and specifically by a given antibody is referred to as an antigen. When a transition-state analog is used as an antigen to stimulate the production of antibodies, the antibodies that bind it are potential catalysts of the corresponding reaction. This approach, first suggested by William P. Jencks in 1969, has become practical with the development of laboratory techniques to produce antibodies that are all identical and bind one specific antigen (these are known as monoclonal antibodies; see Chapter 6).
Pioneering work in the laboratories of Richard Lerner and Peter Schultz has resulted in the isolation of a number of monoclonal antibodies that catalyze the hydrolysis of esters or carbonates (Fig. 2).
In these reactions, the attack by water (OH-) on the carbonyl carbon produces a tetrahedral transition state in which a partial negative charge has developed on the carbonyl oxygen. Phosphonate compounds mimic the structure and charge distribution of this transition state in ester hydrolysis, making them good transition-state analogs; phosphate compounds are used for carbonate reactions.Antibodies that bind the phosphonate or phosphate tightly have been found to catalyze the corresponding ester or carbonate hydrolysis reaction by factors of 103 to 104.Structural analyses of a few of these catalytic antibodies have shown that the catalytic amino acid side chains are arranged where they could interact with the substrate only in the transition state. These studies provide additional evidence for enzyme-transition state complementarity and suggest that new classes of antibody catalysts might be developed for research and industry.
The enzyme reaction of chymotrypsin has two major phases: acylation, in which the peptide bond is cleaved and an ester linkage is formed between the peptide carbonyl carbon and the enzyme; and deacylation, in which the ester linkage is hydrolyzed and the enzyme regenerated. The nucleophile in the acylation phase is the oxygen of Ser195. A serine hydroxyl is normally protonated at neutral pH, but in the enzyme Ser195 is hydrogen-bonded to His57, which is further hydrogen-bonded to Aspl02. These three amino acids are often referred to as a catalytic triad. As the serine oxygen attacks the carbonyl carbon of a peptide bond, the hydrogen-bonded His57 functions as a general base to abstract the serine proton, and the negatively charged Aspl02 stabilizes the positive charge that forms on the His residue. This prevents the development of a very unstable positive charge on the serine hydroxyl and increases its nucleophilicity. His57 can also act as a proton donor to protonate the amino group in the displaced portion of the substrate (the leaving group). A similar set of proton transfers occurs in the deacylation step (Fig. 8-19).
As the serine oxygen attacks the carbonyl group in the substrate, a transition state is reached in which the carbonyl oxygen acquires a negative charge. This charge is formed within a pocket on the enzyme called the oxyanion hole, and it is stabilized by hydrogen bonds contributed by the amide nitrogens of two peptide bonds in the protein backbone. One of these hydrogen bonds occurs only in the transition state and thereby reduces the energy required to reach the transition state. This represents an example of the use of binding energy in catalysis. The importance of binding energy in catalysis by chymotrypsin is discussed further in Box 8-3.
The first evidence for a covalent acyl-enzyme intermediate came from a classic application of pre-steady state kinetics. In addition to its action on polypeptides, chymotrypsin will catalyze the hydrolysis of small ester and amide compounds. These reactions are much slower because less binding energy is available with these substrates, but they are easier to study. Studies by B.S. Hartley and B.A. Kilby found that the hydrolysis of p-nitrophenylacetate by chymotrypsin, as measured by release of p-nitrophenol, proceeded with a rapid burst before leveling off to a slower rate (Fig. 8-20). By extrapolating back to zero time, they concluded that the burst phase corresponded to just under one molecule of p-nitrophenol released for every enzyme molecule present. They suggested that this reflected a rapid acylation of all the enzyme molecules (with release of p-nitrophenol) but that subsequent turnover of the enzyme was limited in rate by a slow deacylation step. Similar results have been obtained with many enzymes. Hexokinase

This is a bisubstrate enzyme (Mr 100,000), catalyzing the interconversion of glucose and ATP with glucose-6-phosphate and ADP. The hydroxyl at position 6 of the glucose molecule (to which the γ-phosphate of ATP is transferred) is similar in chemical reactivity to water, and water freely enters the enzyme active site. Yet hexokinase discriminates between glucose and water, with glucose favored by a factor of
Hexokinase can discriminate between glucose and water because of a conformational change in the enzyme that occurs when the correct substrates are bound (Fig. 8-21). The enzyme thus provides a good example of induced fit. When glucose is not present, the enzyme is in an inactive conformation with the active-site amino acid side chains out of position for reaction. When glucose (but not water) and ATP bind, the binding energy derived from this interaction induces a change to the catalytically active enzyme conformation.
Synthetase This enzyme (Mr 95,000) catalyzes the attachment of tyrosine to an RNA molecule called a transfer RNA, activating the amino acid to form a precursor for protein synthesis (described in Chapter 26). The reaction proceeds in two phases:
Enz + Tyr + ATP ---> Enz •Tyr-AMP + PPi
Enz•Tyr-AMP + tRNA ---> Tyr-tRNA + Enz + AMP
(PPi is the abbreviation for inorganic pyrophosphate. Pi, used later in this chapter, is the abbreviation for inorganic phosphate. ) Kinetic studies have shown that ATP and tyrosine bind to the enzyme in random order. The tyrosyl-AMP intermediate is not released by the enzyme, and is sufficiently stable to allow study of the first reaction phase in isolation. The following discussion focuses on this phase.
The structure of tyrosyl-tRNA synthetase in its complex with tyrosyl-AMP is shown in Figure 8-22. This structure indicates a number of potential hydrogen bonds between enzyme and substrate (Fig. 8-23). Alan Fersht and colleagues have used this information to create, by site-directed mutagenesis (Chapter 28), a series of mutant enzymes lacking one or another of the amino acid side chains contributing to these hydrogen bonds. Hydrogen bonds formed in the ES complex affect Ks, which can be directly measured for this enzyme. Hydrogen bonds formed only in the transition state affect kcat.In several cases, substitution of a nonhydrogen-bonding amino acid at key positions in the active site affectskcat but does not affect Ks. For example, substitution of an alanine for Thr40and a glycine for His45 has little effect on the observed Ks for ATP.
However, these substitutions lower the kcat for the reaction by a factor of 300,000. In other words, this altered enzyme can still bind its substrates to form the ES complex, but once bound, the substrates do not react as rapidly because the enzyme can no longer form two essential hydrogen bonds that normally help to lower the activation energy required to reach the transition state (Fig. 8-23).