These results not only emphasize the capacity of heme FeO + to selectively oxidize a C—H bond as unreactive as a methyl group attached to a saturated aliphatic chain but they also highlight the importance of active site architecture in dictating what part of the substrate molecule is exposed to the oxygen atom of heme FeO3 +. Members of CYP families 1, 2, and 3, as major drug-metabolizing enzymes, would be expected to have relatively open active and less constraining active sites to accommodate a greater variety of molecules. Conversely, members of CYP families, such as CYP4, that are involved in the metabolism of endogenous bioactive molecules, such as the prostaglandins or the steroids, would be expected to have active sites that confine the oxidation of specific molecules to specific sites within the molecule. This indeed appears to be the case.
That nature has demonstrated the possibility of constructing cytochromes P450 that can selectively oxidize sites within complex molecules that are neither energetically nor statistically favored suggests that it may be possible to bioengineer cytochromes P450 to obtain catalysts for the production of important intermediates or end products that can only be obtained with great difficulty or at great expense. Catalysts could also be engineered for processing recalcitrant environmental pollutants such as polychlorinated aromatic compounds (e.g., dioxin). Such possibilities have provoked a great deal of interest within the scientific community, and their exploration is beginning to meet with some success. While a thorough discussion of the area is beyond the scope of this chapter, a few examples are worth noting.
Fisher etal.37 covalently linked rat cytochrome P450 reductase to rat CYP4A1, a cytochrome P450 responsible for the o-hydroxylation of lauric acid (5) (eqn ). The new fusion protein is extremely active, and catalyzes this reaction at the remarkable turnover rate of 300nmol(nmol P450) _ 1 min_ 1.38 The exceptional rate is presumably achieved by compelling the reductase and cytochrome P450 to maintain close proximity by covalently linking the two proteins. Thus, the price of entropy is paid as the reductase is always in position to supply reducing equivalents to the cytochrome P450 and fuel the reaction.
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