Enzyme-substrate transition state complexes

Efficiency and selectivity are the two keywords that better outline the outstanding performances of enzymes. However, in some cases unsatisfactory stereoselectivity of enzymes can be found and, in these cases, the enantiomeric excesses of products are too low for synthetic purposes. In order to overcome this limitation, a number of techniques have been proposed to enhance the selectivity of a given biocatalyst. The net effect pursued by all these protocols is the increase of the difference in activation energy (AAG ) of the two competing diastereomeric enzyme-substrate transition state complexes (Figure 1.1). 

The function of enzymes is to accelerate the rates of reaction for specific chemical species. Enzyme catalysis can be understood by viewing the reaction pathway, or catalytic cycle, in terms of a sequential series of specific enzyme-ligand complexes (as illustrated in Figure 1.6), with formation of the enzyme-substrate transition state complex being of paramount importance for both the speed and reactant fidelity that typifies enzyme catalysis. 

There are important consequences for this statement. The enzyme must stabilize the transition-state complex, EX, more than it stabilizes the substrate complex, ES. Put another way, enzymes are designed by nature to bind the transition-state structure more tightly than the substrate (or the product). The dissociation constant for the enzyme-substrate complex is... 

Thus, the enzymatic rate acceleration is approximately equal to the ratio of the dissociation constants of the enzyme-substrate and enzyme-transition-state complexes, at least when E is saturated with S. 

We have just discussed several common strategies that enzymes can use to stabilize the transition state of chemical reactions. These strategies are most often used in concert with one another to lead to optimal stabilization of the binary enzyme-transition state complex. What is most critical to our discussion is the fact that the structures of enzyme active sites have evolved to best stabilize the reaction transition state over other structural forms of the reactant and product molecules. That is, the active-site structure (in terms of shape and electronics) is most complementary to the structure of the substrate in its transition state, as opposed to its ground state structure. One would thus expect that enzyme active sites would bind substrate transition state species with much greater affinity than the ground state substrate molecule. This expectation is consistent with transition state theory as applied to enzymatic catalysis. 

CATALYSIS. Any condition promoting formation will tend to speed up the reaction rate, and catalysts are thought to accomplish rate enhancement chiefly by stabilizing the transition state. Shown in Fig. 8 is an enzyme-catalyzed process in which reactant S (more commonly called substrate in enzymology) combines with enzyme to form an enzyme-substrate complex. This complex leads to formation of the transition state complex EX which may proceed to form enzyme-product complex. The catalytic reaction cycle is then completed by the release of product P, whereupon the uncombined enzyme returns to its original state. 

Schematic representation of energy changes accompanying formation of enzyme-substrate complex and subsequent formation of a transition-state complex.

Because in catalysis the enzyme-substrate complex is destabilized and the energy so involved is released on forming the transition state, the enzyme binds the substrate very tightly in the transition state. Some enzymes can be dramatically inhibited by so-called transition-state analogs. The transition state normally has only a fleeting existence (<1013 s), but the analogs are stable structures that resemble the postulated transition-state complex. 

Calculate the rate enhancement that would be achieved if the activation energy of the transition-state complex of an enzyme with its substrate were halved. 

Since a large number of xenobiotics are metabolized to free radicals, an overall view of this area is not obvious. By definition, free radical metabolites must exist free of the enzyme, and, therefore, enzyme-xenobiotic transition states with free radical character such as are thought to exist in the cytochrome P-450 substrate complex are excluded. It follows that if the rate of formation of the free radical is fast enough, it can be detected with electron spin resonance, and will have the same ESR spectrum as the free radical made by purely chemical means. 

The favoured model of enzyme-substrate interaction is referred to as the induced fit model (Figure 9.1). An initial interaction between enzyme and substrate induces a conformational change in the protein that strengthens further binding and brings the catalytic site close to substrate bonds to be altered, generating transition-state complexes and reaction products. 

Schematic representation of the proposed mechanism for the transfer of GIcNAc catalyzed by GIcNAc-TI. ES, enzyme-substrate complex TS, transition state complex PC, product complex...

Enzyme kinetics is the quantitative study of enzyme catalysis. According to the Michaelis-Menten model, when the substrate S binds in the acdve site of an enzyme E, an ES transition state complex is formed. During the transition state, the substrate is converted into product. After a time the product dissociates from the enzyme. In the Michaelis-Menten equation,... 

Many biological catalysts, enzymes, often acquire their catalytic functions under these stringent reaction conditions by incorporating transition metals into their catalytic site. The metals are coordinated to ligands, which are constituents of the polypeptide chain and participate in the formation of transition-state complexes with substrates in the performance of biochemical reactions. Whereas Nature can provide a rich diversity of organometallic catalysts that require an aqueous solvent, it is a challenge - as seen below - to the chemist to duplicate these enzymic reactions so that they can be exploited and adapted to industrial-scale processes. 

A further point that emerges from perturbation models is an interpretation of enzymatic reactions. If one says that binding energy is utilized to lower the energy of the transition state , it is implied that the transition state binds to the enzyme better than the ground state does. In terms of our model, the enzyme may be considered as perturbing the structure of the substrate in the enzyme-substrate groundstate complex in the direction of the transition-state complex. Compared to the uncatalyzed or unperturbed reaction, the effects on reaction rate can be profound (see Chapter 13). 

Fig. 4. Schematic showing the rate-limiting acylation step in the hydrolysis of peptide bonds by subtilisin. In going from the Michaelis enzyme-substrate complex (E-S) to the transition state complex (E-S ), the proton on Ser-221 (darkly shaded) is transferred to His-64, thus permitting nucleophilic attack on the scissile peptide bond. [Reprinted with permission from Ref. (46).J...

It therefore seems that the very same active site can offer numerous modes of interactions, and some of these might be utilized by promiscuous substrates. It should be noted, however, that most of the above describes cases analyzed by kinetics and site-directed mutagenesis. Very few structures of the enzyme—substrate, or enzyme transition-state complexes, exist for both the native and promiscuous substrates. And thus, small, or even significant, changes in active-site configuration cannot be excluded in the described cases. 

Fig. 6. Standard state free-energy changes for a reaction with the same transition state in the absence and presence of enzyme. Hypothetical case where the enzyme-substrate (in the ground state) and enzyme-transition state complexes are equally stabilised. There can be no catalysis either above saturation (a) or below saturation (b).

Fig. 8.4. Reaction in the enzyme active catalytic site. A. The enzyme contains an active catalytic site, shown in dark blue, with a region or domain where the substrate binds. The active site also may contain cofactors, nonprotein components that assist in catalysis. B. The substrate forms bonds with amino acid residues in the substrate binding site, shown in light blue. Substrate binding induces a conformational change in the active site. C. Functional groups of amino acid residues and cofactors in the active site participate in forming the transition state complex, which is stabilized by additional noncovalent bonds with the enzyme, shown in blue. D. As the products of the reaction dissociate, the enzyme returns to its original conformation.

The transition state complex decomposes to prodncts, which dissociate from the enzyme (see Fig. 8.4D). The enzyme generally returns to its original form. The free enzyme then binds another set of substrates, and repeats the process. 

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