The catalytic site of cytochrome P4504A11 (CYP4A11) and its L131F mutant.
CYP4A11, the principal known human fatty acid omega-hydroxylase, has been expressed as a polyhistidine-tagged protein and purified to homogeneity. Based on an alignment with P450BM-3, the CYP4A11 L131F mutant has been constructed and similarly expressed. The two proteins are spectroscopically indistinguishable, but wild-type CYP4A11 primarily catalyzes omega-hydroxylation, and the L131F mutant only omega-1 hydroxylation, of lauric acid. The L131F mutant is highly uncoupled in that it slowly (omega-1)-hydroxylates lauric acid yet consumes NADPH at approximately the same rate as the wild-type enzyme. Wild-type CYP4A11 is inactivated by 1-aminobenzotriazole under turnover conditions but the L131F mutant is not. This observation, in conjunction with the binding affinities of substituted imidazoles for the two proteins, indicates that the L131F mutation decreases access of exogenous substrates to the heme site. Leu-131 thus plays a key role in controlling the regioselectivity of substrate hydroxylation and the extent of coupled versus uncoupled enzyme turnover. A further important finding is that the substituted 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 the presence of cytochrome P450 reductase and NADPH.
Dierks
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Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0446, USA.
Zhang
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Johnson
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de Montellano
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001665:Binding Sites; D042926:Cytochrome P-450 CYP4A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D015525:Fatty Acids, Omega-3 / Q000096:biosynthesis; D006801:Humans; D007093:Imidazoles / Q000378:metabolism; D007097:Imines / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009251:NADPH-Ferrihemoprotein Reductase; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism; D014230:Triazoles / Q000494:pharmacology
D001426:Bacterial Proteins; D065607:Cytochrome P-450 Enzyme Inhibitors; D015525:Fatty Acids, Omega-3; D007093:Imidazoles; D007097:Imines; D011994:Recombinant Proteins; D014230:Triazoles; C033020:1-aminobenzotriazole; D003577:Cytochrome P-450 Enzyme System; C011981:phenyldiazene; D006899:Mixed Function Oxygenases; D042926:Cytochrome P-450 CYP4A; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases