THE CATALYTIC SITE OF CYTOCHROME P4504A11 (CYP4A11) AND ITS L131F MUTANT

CYP4A11, the principal known human fatty acid omega-hydroxylase, has b een expressed as a polyhistidine-tagged protein and purified to homoge neity, Based on an alignment with P450(BM-3), the CYP4A11 L131F mutant has been constructed and similarly expressed. The two proteins are sp ectroscopically indistinguishable, but wildtype CYP4A11 primarily cata lyzes omega-hydroxylation, and the L131F mutant only omega-1 hydroxyla tion, of lauric acid. The L131F mutant is highly uncoupled in that it slowly (omega-1)-hydroxylates lauric acid yet consumes NADPH at approx imately the same rate as the wild-type enzyme. Wild-type CYP4A11 is in activated by 1-aminobenzotriazole under turnover conditions but the L1 31F mutant is not. This observation, in conjunction with the binding a ffinities of substituted imidazoles for the two proteins, indicates th at the L131F mutation decreases access of exogenous substrates to the heme site. Leu-131 thus plays a key role in controlling the regioselec tivity of substrate hydroxylation and the extent of coupled versus unc oupled enzyme turnover. A further important finding is that the substi tuted imidazoles bind more weakly to CYP4A11 and its L131F mutant when these proteins are reduced by NADPH-cytochrome P450 reductase than by dithionite. This finding suggests that the ferric enzyme undergoes a conformational change that depends on both reduction of the iron and t he presence of cytochrome P450 reductase and NADPH.