A SINGLE MUTATION IN CYTOCHROME-P450 BM3 CHANGES SUBSTRATE ORIENTATION IN A CATALYTIC INTERMEDIATE AND THE REGIOSPECIFICITY OF HYDROXYLATION

Phenylalanine 87 of Bacillus megaterium cytochrome P450 BM3, a residue close to the heme in the substrate binding pocket, has been replaced by alanine by site-directed mutagenesis. The substitution had no effec t on the rate of hydroxylation of laurate and increased the affinity f or laurate of both the intact enzyme and its heme domain by 2.6-6-fold in the ferric state. NMR paramagnetic relaxation measurements showed that in the initial ferric enzyme-substrate complex, where the substra te binds relatively far from the heme, the substitution had no effect on the position or orientation of the bound substrate. However, in the next intermediate in the catalytic cycle, the reduced enzyme, the pos ition of the bound substrate was altered so that the terminal methyl g roup was 3.1 Angstrom from the iron in the mutant, compared to 5.1 Ang strom in the wild-type enzyme. Analysis of the products of the action of the enzyme on laurate and myristate showed-that the mutant catalyze d hydroxylation almost exclusively at the omega position, in marked co ntrast to the wild-type enzyme, with which no hydroxylation at this po sition was observed.