Enzyme-substrate transition state

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. 

According to transition-state theory the second-order rate constant k yK is directly related to the free-energy difference (AG ) between the enzyme-substrate transition state (ES ) and the/ree unbound substrate and enzyme (Eqn. 20) by Eqn. 

The second group of studies tries to explain the solvent effects on enantioselectivity by means of the contribution of substrate solvation to the energetics of the reaction [38], For instance, a theoretical model based on the thermodynamics of substrate solvation was developed [39]. However, this model, based on the determination of the desolvated portion of the substrate transition state by molecular modeling and on the calculation of the activity coefficient by UNIFAC, gave contradictory results. In fact, it was successful in predicting solvent effects on the enantio- and prochiral selectivity of y-chymotrypsin with racemic 3-hydroxy-2-phenylpropionate and 2-substituted 1,3-propanediols [39], whereas it failed in the case of subtilisin and racemic sec-phenetyl alcohol and traws-sobrerol [40]. That substrate solvation by the solvent can contribute to enzyme enantioselectivity was also claimed in the case of subtilisin-catalyzed resolution of secondary alcohols [41]. 

Figure 1.6 Schematic representation of the changes in protein conformational microstate distribution that attend ligand (i.e., substrate, transition state, product and inhibitor) binding during enzyme catalysis. For each step of the reaction cycle, the distribution of conformational microstates is represented as a potential energy (PE) diagram.

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. 

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. 

If enzymes bind transition states more tightly than substrates, then they should bind molecules which resemble transition states in shape and/or charge more tightly than they bind substrates or products. The concept of transition state analogue was first applied to glycosyl transfer by Leaback, " who... 

Enzymes are biological catalysts. They enhance reaction rates because they provide an alternative reaction pathway that requires less energy than an uncatalyzed reaction. In contrast to some inorganic catalysts, most enzymes catalyze reactions at mild temperatures. In addition, enzymes are specific to the types of reactions they catalyze. Each type of enzyme has a unique, intricately shaped binding surface called an active site. Substrate binds to the enzyme s active site, which is a small cleft or crevice in a large protein molecule. In the lock-and-key model of enzyme action, the structures of the enzyme s active site and the substrate transition state are complementary. In the induced-fit model, the protein molecule is assumed to be flexible. 

Distortion of substrate/transition state stabilization In model reactions, it was demonstrated that distortion of a reactant so as to make it more closely resemble the postulated transition state of the reaction can lead to dramatic acceleration. In a number of enzyme-catalyzed reactions, there is evidence for such strain or... 

---