Enzyme transition state stabilization

Transition-state stabilization in chymotrypsin also involves the side chains of the substrate. The side chain of the departing amine product forms stronger interactions with the enzyme upon formation of the tetrahedral intermediate. When the tetrahedral intermediate breaks down (Figure 16.24d and e), steric repulsion between the product amine group and the carbonyl group of the acyl-enzyme intermediate leads to departure of the amine product. 

The differences in the rate constant for the water reaction and the catalyzed reactions reside in the mole fraction of substrate present as near attack conformers (NACs).171 These results and knowledge of the importance of transition-state stabilization in other cases support a proposal that enzymes utilize both NAC and transition-state stabilization in the mix required for the most efficient catalysis. Using a combined QM/MM Monte Carlo/free-energy perturbation (MC/FEP) method, 82%, 57%, and 1% of chorismate conformers were found to be NAC structures (NACs) in water, methanol, and the gas phase, respectively.172 The fact that the reaction occurred faster in water than in methanol was attributed to greater stabilization of the TS in water by specific interactions with first-shell solvent molecules. The Claisen rearrangements of chorismate in water and at the active site of E. coli chorismate mutase have been compared.173 It follows that the efficiency of formation of NAC (7.8 kcal/mol) at the active site provides approximately 90% of the kinetic advantage of the enzymatic reaction as compared with the water reaction. 

The structural and chemical mechanisms used by enzymes to achieve transition state stabilization have been reviewed in detail elsewhere (e.g., see Jencks, 1969, Warshel, 1998, Cannon and Benkovic, 1998, Copeland, 2000, Copeland and Anderson, 2002 and Kraut et al., 2003). Four of the most common strategies used by enzymes for transition state stabilization—approximation, covalent catalysis, acid/base catalysis, and conformational distortion—are discussed below. 

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. 

In this chapter we have seen that enzymatic catalysis is initiated by the reversible interactions of a substrate molecule with the active site of the enzyme to form a non-covalent binary complex. The chemical transformation of the substrate to the product molecule occurs within the context of the enzyme active site subsequent to initial complex formation. We saw that the enormous rate enhancements for enzyme-catalyzed reactions are the result of specific mechanisms that enzymes use to achieve large reductions in the energy of activation associated with attainment of the reaction transition state structure. Stabilization of the reaction transition state in the context of the enzymatic reaction is the key contributor to both enzymatic rate enhancement and substrate specificity. We described several chemical strategies by which enzymes achieve this transition state stabilization. We also saw in this chapter that enzyme reactions are most commonly studied by following the kinetics of these reactions under steady state conditions. We defined three kinetic constants—kai KM, and kcJKM—that can be used to define the efficiency of enzymatic catalysis, and each reports on different portions of the enzymatic reaction pathway. Perturbations... 

Wild-type and mutant enzymes Main a-linkage in glucan products Amino acid sequence around transition-state stabilizer D627 in GTFR... 

At the end of the review there are some examples involving catalysis by acids and bases, metal ions, micelles, amylose, catalytic antibodies, and enzymes to give the reader a feeling for how Kurz s approach may be usefully applied to other catalysts. Very few of these examples, or those involving cyclodextrins, were discussed in the original literature in the same terms. It is hoped that the present treatment will stimulate further use and exploration of the Kurz approach to analysing transition state stabilization. 

As impressive as these developments have been, chemists still have a way to go to catch up with Mother Nature . For enzymes KTS may be as low as 10 2( m, since is generally in the range 10-3 to IO m and kjku values are up to 1014 or more (Lienhard, 1973 Kraut, 1988) (see Enzymes, Section 6). Further lowering of KTS for artificial enzymes below 10 ,om will no doubt require more covalent interactions in the transition state, with better catalytic groups. Nevertheless, the transition state stabilization evident in Table 4 is comparable to that which has been achieved so far with catalytic antibodies (Section 6). 

Enzymes have evolved their awesome efficiency over billions of years. Mankind does not have that much time In developing new, highly selective, and possibly totally synthetic catalysts, we must use whatever theoretical, practical, and heuristic tools are available to us. The concept of transition state stabilization is one such tool. 

Is the transition state stabilized more than the ground states due to extra bonding from the enzyme ... 

The crystal structure of the cobalt-substituted enzyme was obtained with bicarbonate bound to the metal (Iverson et al. 2000). The structure shows Asn 202 and Gln75 hydrogen bonded to the metal-bound bicarbonate, suggestzing potential roles for these residues in either transition-state stabilization or orientation and polarization of CO2 for attack from the zinc-hydroxyl (Fig. 11.5). The crystal structure also shows three discrete conformations for Glu 84, suggesting a role for this residue in the transfer of protons out of the active site indeed, kinetic analyses of Glu 84 variants combined with chemical rescue experiments establish this residue as critical for proton transfer (Tripp and Ferry 2000). The location of Glu 62 adjacent to Glu 84 suggests a potential role in proton transfer as well. Although kinetic analyses of site-specific variants establish an essential role for Glu 62 in the CO2 hydration steps (Eqs. 11.3 and 11.4), the results were inconclusive regarding an additional role in proton transfer (Eqs. 11.5 and 11.6). 

Investigations of enzyme-catalyzed direct electron transfer introduce the basis for a future generation of electrocatalysts based on enzyme mimics. This avenue could offer new methods of synthesis for nonprecious metal electrocatalysts, based on nano-structured (for example, sol—gel-derived) molecular imprints from a biological catalyst (enzyme) with pronounced and, in some cases, unique electrocatalytic properties. Computational approaches to the study of transition state stabilization by biocatalysts has led to the concept of theozymes . " ... 

Bovine pancreatic chymotrypsin (Mr 25,191) is a protease, an enzyme that catalyzes the hydrolytic cleavage of peptide bonds. This protease is specific for peptide bonds adjacent to aromatic amino acid residues (Trp, Phe, Tyr). The three-dimensional structure of chymotrypsin is shown in Figure 6-18, with functional groups in the active site emphasized. The reaction catalyzed by this enzyme illustrates the principle of transition-state stabilization and also provides a classic example of general acid-base catalysis and covalent catalysis. 

Transition-state stabilization The active site often acts as a flexible molecular template that binds the substrate in a geometric structure resembling the activated transition state of the molecule (see T at the top of the curve in Figure 5.4). By stabilizing the substrate in its transition state, the enzyme greatly increases the concentration of the reactive intermediate that can be converted to product and, thus, accelerates the reaction. 

Strain (see also transition state stabilization) 369-374 equilibria on enzyme surface and 118... 

The situation in which the enzyme has a group that can bind to the substrate only in the transition state is an example of transition state stabilization. It is important to note that the presence on the enzyme of a group that can bind only to the transition state of the substrate decreases the activation energy for a chemical step, and, further, does not have to distort the substrate to do so. 

Strain and stress in enzymes arise from several different causes. We have seen in this chapter, and we shall see further in Chapters 15 and 16, that stress and strain may be divided into two processes, substrate destabilization and transition state stabilization. Substrate destabilization may consist of steric strain, where there are unfavorable interactions between the enzyme and the substrate (e.g., with proline racemase, lysozyme) desolvation of the enzyme (e.g., by displacement of two bound water molecules from the carboxylate of Asp-52 of lysozyme) and desolvation of the substrate (e.g., by displacement of any bound water molecules from a peptide28). Transition state stabilization may consist of the presence of transition state binding modes that are not available for the... 

The transition state-stabilizing power of the rapid enzymic process can be held up for comparison with a particularly slow nonenzymic reaction that is brought about through addition of a large amount of heating. In particular, comparisons of the rates of enzyme-catalyzed process and spontaneous nonenzymic reaction produce dramatically large ratios. 

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