He You Ai, Roussel Fabienne, Halpert James R
Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston 77555-1031, USA.
Arch Biochem Biophys. 2003 Jan 1;409(1):92-101. doi: 10.1016/s0003-9861(02)00484-8.
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 microM) than to free enzyme (K(S1)=960 microM). 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.
已对人细胞色素P450 3A4（CYP3A4）及其40个突变体催化地西泮氧化的协同作用的结构基础进行了研究。采用一种有序双位点模型来解释替马西泮形成的S形动力学以及去甲地西泮形成的双曲线动力学，在该模型中，底物首先结合到催化/效应位点，然后再结合到催化位点。在该模型中，地西泮与酶-底物复合物的结合亲和力（K(S2)=140 microM）高于与游离酶的结合亲和力（K(S1)=960 microM）。残基107、119、211、301、304、309、369、370和373在决定地西泮氧化的区域选择性方面发挥重要作用。有趣的是，S119F和A370F在去甲地西泮形成方面表现出S形动力学，而I301F对两种产物均表现出双曲线动力学。在睾酮浓度增加的情况下，地西泮的K(S1)降低，而K(S2)增加。数据表明活性口袋内存在三个位点。
