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638 Enzyme Mechanisms

FIGURE 9 The cytochrome P450 cam structure. The bound heme is depicted in black, and the iron atom at the center
of the heme appears as a sphere. [Adapted from Poulos, T. L., Finzel, B. C., and Howard, A. J. (1986). “Crystal
structure of substrate-free Pseudomonas putida cytochrome P450,” Biochemistry 25, 5314–5322.]

is then displaced by water; this completes the catalytic the question of “how enzymes work” cannot be described
cycle. fully in a single scheme.
One important line of investigation which has supported
the radical rebound hypothesis is the use of radical clock
substrate probes. These probes rearrange in a diagnostic IV. ORIGINS OF THE CATALYTIC
way on a very rapid and calibrated time scale when a EFFICIENCY OF ENZYMES
hydrocarbon radical is formed. In the case of P450, re-
arranged products have been isolated after oxidation and The source of the stereospecificity of enzyme-catalyzed
have been used as evidence of an intermediate substrate reactions is clearly revealed by the fit of the substrate to the
radical. In this way, even though the lifetime of the radical enzyme’s active site that spatially then directs the stere-
is too short for it to be observed directly, its character can ochemical course of the chemical events. The speed of
be explored by the judicious choice of substrate analogues. these reactions has been attributed to the lowering of the
Mechanistic proposals are under constant scrutiny and activation energy for the process by the greater affinity of
revision, and aspects of the foregoing mechanism have the enzyme for the transition state than that for the sub-
been challenged. In particular, the possibility has been strate. Although this proposal is an adequate rationale, it
suggested that a species other than a high valent iron-oxo is often a necessary thermodynamic statement that does
(likely a hydroperoxo species) may be the active oxidant not offer insights into how the activation barrier is actually
for some substrates. Debates such as these are a great lowered.
strength of the study of enzyme mechanisms. Given all The preorganization of substrate and active site residues
the tools which have been developed in this field, and the within a protein cavity converts an intermolecular pro-
wealth of interesting problems to which these tools can cess to intramolecular and may have both an enthalpic
be applied, the study of enzyme mechanisms should be and an entropic advantage. The active site provides an
considered a vital and evolving process. The answer to environment in which the enzyme·substrate complex is