Role of residue 478 as a determinant of the substrate specificity of cytochrome P450 2B1.
Two allelic variants and eight site-directed mutants of cytochrome P450 2B1 differing at residue 478 have been expressed in COS cells and assayed for androstenedione hydroxylase activities. The 478Gly and 478Ala variants and five mutants (Ser, Thr, Val, Ile, and Leu) exhibited 16 beta-OH:16 alpha-OH ratios ranging from 0.7 to 9.3, whereas the Pro, Glu, and Arg mutants were expressed but inactive. The seven samples active toward androstenedione also exhibited testosterone 16 beta-OH:16 alpha-OH ratios ranging from 0.4 to 2.3. With both steroids, the Gly variant had the highest 16 beta-hydroxylase activity, and the 16 beta-OH:16 alpha-OH ratio increased with the size of aliphatic size chains (Ala, Val, and Ile/Leu). The highest ratio of androgen 15 alpha:16-hydroxylation was observed with the Ser mutant. On the basis of previous work indicating decreased susceptibility of the 478Ala variant in liver microsomal and reconstituted systems to inactivation by chloramphenicol analogs, methodology was refined for monitoring enzyme inactivation in COS cell microsomes. The Gly and Ala variants were inactivated by chloramphenicol with similar rate constants, whereas the Ser and Val mutants were inactivated more slowly, and the Leu mutant was refractory. Only the Gly variant was inactivated by the chloramphenicol analog N-(2-p-nitrophenethyl)chlorofluoroacetamide. Thus, the side chain of residue 478 appears to be a major determinant of enzyme inactivation as well as of androgen hydroxylation.(ABSTRACT TRUNCATED AT 250 WORDS)
He
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Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson 85721.
Balfour
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Kedzie
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Halpert
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JR
D000483:Alleles; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D002460:Cell Line; D002701:Chloramphenicol / Q000031:analogs & derivatives / Q000494:pharmacology; D019362:Cytochrome P-450 CYP2B1; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014644:Genetic Variation; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013250:Steroid Hydroxylases / Q000378:metabolism*; D013379:Substrate Specificity; D014162:Transfection
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D002701:Chloramphenicol; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D019362:Cytochrome P-450 CYP2B1; C050356:steroid 15-alpha-hydroxylase; C022724:testosterone 7-alpha-hydroxylase, hamster