ORIGINS OF THE CATALYTIC EFFICIENCY OF ENZYMES

The source of the stereospecificity of enzyme-catalyzed reactions is clearly revealed by the fit of the substrate to the enzyme’s active site that spatially then directs the stereochemical course of the chemical events. The speed of these reactions has been attributed to the lowering of the activation energy for the process by the greater affinity of the enzyme for the transition state than that for the substrate. Although this proposal is an adequate rationale, it is often a necessary thermodynamic statement that does not offer insights into how the activation barrier is actually lowered.

The preorganization of substrate and active site residues within a protein cavity converts an intermolecular process to intramolecular and may have both an enthalpic and an entropic advantage. The active site provides an environment in which the enzyme-substrate complex is populated with cofactors that are poised for reaction. These structures, or NACs (near attack conformers), are similar in structure to the transition state so that only slight changes in bond distances and angles within the structures through the normal dynamic motions of the protein are sufficient to trigger the crossing of the reaction barrier. The enzyme’s active site is also preorganized in the sense that the locus of general acids/bases, nucleophiles, solvents, dipoles, hydrogen bonds, and so forth are fixed by the NAC to interact with the transition state. Molecular dynamic calculations sampling several enzyme classes suggest that the affinity of these enzymes for their transition states is little changed from that for the substrate. The enzyme-catalyzed reaction also benefits in many cases due to the nonaqueous interaction of the active site cavity, which can often accelerate, by large factors, the reaction over that in aqueous media.

The cytochrome P450cam structure. The bound heme is depicted in black, and the iron atom at the center of the heme appears as a sphere.

FIGURE 9 The cytochrome P450cam structure. The bound heme is depicted in black, and the iron atom at the center of the heme appears as a sphere.

A currently accepted version of the catalytic cycle of cytochrome P450. The iron porphyrin is drawn as a parallelogram, the substrate is designated as SH and the product as SOH.

FIGURE 10 A currently accepted version of the catalytic cycle of cytochrome P450. The iron porphyrin is drawn as a parallelogram, the substrate is designated as SH and the product as SOH.

For DHFR in particular, molecular dynamics calculations, NMR measurements of solution structure, and kinetics measurements of mutant forms of the enzyme appear to support the importance of dynamic motions of the protein fold to trigger the reaction of an enzyme-substrate NAC. The mutations in question (for example Gly120 in Fig. 7) are well removed from the active site and underscore the role of the entire protein fold. The contribution of dynamic motions to the overall catalytic rate remains to be elucidated for the majority of enzymes. Their existence may explain why more rigid molecules such as imprinted polymers and catalytic antibodies do not generally exhibit the large rate accelerations noted with enzymes despite the fact that they too have converted an intermolecular process to an intramolecular process.

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