Mutations of phenylalanine-193 in the putative substrate access channel alter the substrate specificity of cytochrome P450(cam)

The Phe-193 residue on the surface of cytochrome P450(cam) is part of a clu ster of residues proposed to undergo dynamic fluctuations to permit the ent ry of substrates into the active site pocket. The role of this residue in t he activity of P450(cam) has been investigated. The F193A, F193V, F1931, an d F193L mutations were introduced into the Y96F mutant, which had been show n to oxidize a wider range of molecules at faster rates than the wild-type enzyme. The F193L mutation had very little effect, while the F193A and F193 I mutations reduced the camphor oxidation rate and almost abolished the sty rene and naphthalene oxidation activity of the Y96F mutant. In contrast, th e high activity of the Y96F mutant for the oxidation of adamantane, hexane, and 3-methylpentane was largely retained, although the product distributio ns were significantly altered. This dramatic difference between the F193L a nd F193I mutations warrants further investigation. The turnover rates of th e Y96F-F193I with all the substrates showed the same dependence on the Pd:P 450(cam) concentration ratio as for the Y96F mutant, clearly indicating tha t if the F193 mutations had affected substrate access, substrate entry was still fast compared to the first electron transfer, which remained the rate -limiting step for the overall reaction. We concluded that the F193A and F1 93I mutations shifted the substrate specificity of P450(cam) by causing str uctural changes that were relayed from their surface position down to the v icinity of the heme. The altered substrate binding resulted in differential electron transfer kinetics between classes of compounds.