Journal
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Volume 409, Issue 1, Pages 92-101Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/S0003-9861(02)00484-8
Keywords
cytochrome P450 3A4; CYP3A4; diazepam; testosterone; kinetics; sigmoidal and hyperbolic curves; homotropic and heterotropic cooperativity; site-directed mutagenesis
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Funding
- NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES [P30ES006676] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R01GM054995, R37GM054995] Funding Source: NIH RePORTER
- NIEHS NIH HHS [ES06676] Funding Source: Medline
- NIGMS NIH HHS [GM54995] Funding Source: Medline
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The structural basis for the cooperativity of diazepam oxidation catalyzed by human cytochrome P450 3A4 (CYP3A4) and 40 mutants has been investigated. An ordered two-site model in which substrates bind first to a catalytic/effector site and then to the catalytic site was used to explain sigmoidal kinetics for temazepam formation but hyperbolic kinetics for nordiazepam formation. In this model diazepam binds to the enzyme-substrate complex with a greater affinity (K-S2 = 140 muM) than to free enzyme (K-S1 = 960 muM). Residues 107, 119, 211, 301, 304, 309, 369, 370, and 373 play an important role in determining regioselectivity of diazepam oxidation. Interestingly, S119F and A370F displayed sigmoidal kinetics for nordiazepam formation, whereas I301F exhibited hyperbolic kinetics for both products. In the presence of increasing concentrations of testosterone, K-S1 for diazepam decreased, whereas K-S2 increased. The data suggest that three sites exist within the active pocket. (C) 2002 Elsevier Science (USA). All rights reserved.
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