MUTAGENESIS AT A HIGHLY CONSERVED PHENYLALANINE IN CYTOCHROME-P450 2E1 AFFECTS HEME INCORPORATION AND CATALYTIC ACTIVITY

The phenylalanine corresponding to Phe-429 of rabbit cytochrome P450 2E1 is 1 of approximately 10 highly conserved residues in this superfamily of over 200 sequenced enzymes. This nearly invariant residue has been postulated to be involved in electron transfer between the heme of cytochrome P450(cam) and its redox partners [Stayton, P. S., Poulos, T. L., and Sligar, S. G. (1989) Biochemistry 28, 8201-8205]. To test this hypothesis, oligonucleotide-directed mutagenesis was used to replace this amino acid in rabbit P450 2E1 with aspartate, arginine, leucine, tryptophan, or tyrosine, and the mutant proteins were expressed in Escherichia coli. Although immunoblot analysis of whole cell lysates demonstrated that all P450 proteins (mutants and wild-type) were equally well expressed on a per cell basis, in solubilized membranes only the tryptophan and tyrosine mutants yielded ferrous-CO difference spectra characteristic of P450. The specific content (nanomoles per milligram of membrane protein) and yield per liter of the Trp mutant holoenzyme were approximately one-third those of the native enzyme, suggesting that heme incorporation was hindered by tryptophan at this position, whereas the specific content and yield per liter of the Tyr mutant were significantly greater than those of the native preparation. The stability of the Trp and Tyr mutants, as judged by thermal denaturation studies, was not different from that of the native enzyme. The Trp mutant had 38% of the aniline hydroxylase activity, 25% of the p-nitrophenol hydroxylase activity, and 39% of the N-nitrosodimethylamine demethylase activity of the native enzyme, demonstrating that this substitution also decreased catalytic activity. In contrast, substitution of tyrosine at this position had no effect on these activities. The K-m value of aniline was not significantly affected by either substitution, suggesting that these two mutations caused minimal perturbation to the substrate binding site. NADPH oxidation in both the presence and absence of substrate was significantly reduced with the tryptophan mutant, suggesting that electron flow between the reductase and P450 was impaired by this mutation. These results indicate that substitutions at this position can alter electron flow between the reductase and P450 and that this may be the mechanism by which the catalytic rate is reduced with the tryptophan mutant.