Adams J D, Yagi H, Levin W, Jerina D M
University of Southern California, School of Pharmacy, Los Angeles 90033.
Chem Biol Interact. 1995 Mar 30;95(1-2):57-77. doi: 10.1016/0009-2797(94)03354-4.
The active site of cytochrome P450 1A1 has been probed with the substrate 7,8-dihydrobenzo[a]pyrene using a purified, reconstituted system composed of cytochrome P450 1A1, NADPH-cytochrome c reductase and lipid in the presence or absence of epoxide hydrolase. The turnover of the substrate was found to be 38 nmol/nmol of cytochrome P450/min. The metabolic products that were identified are: a phenolic 7,8-dihydrobenzo[a]pyrene (20-29%); 9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (17-28%); benzo[a]pyrene (12-19%); 7-hydroxy-7,8-dihydrobenzo[a]pyrene (13-16%); 8-hydroxy-7,8-dihydrobenzo[a]pyrene (7-15%); 3-hydroxybenzo[a]pyrene (7-15%); 4,5-epoxy-4,5,7,8-tetrahydrobenzo[a]pyrene (0-4%); and a triol of 7,8,9,10-tetrahydrobenzo[a]pyrene (0-4%). 9,10-Epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene undergoes rapid hydrolysis to cis- and trans-9,10-dihydroxy-dihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (2:1) by benzylic attack of water at C-10. Approximately 71% of the trans diols are derived from (+)-(9S,10R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, indicating that cytochrome P450 1A1 has more than a 2:1 preference for selective epoxidation of an enantiotopic face of 7,8-dihydrobenzo[a]pyrene. This stereo-selectivity agrees with the postulated stereo-selectivity predicted by a previously described active site model for cytochrome P450 1A1. Epoxide hydrolase in pure form or in hepatic microsomes catalyzes the hydrolysis of 9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, which is inhibited by 1,1,1-trichloropropane 2,3-oxide. The (+)-(9S,10R)-isomer of the epoxide is slightly preferred as a substrate over its enantiomer and is cleaved by benzylic and nonbenzylic attack. Only benzylic attack was found with (-)-(9R,10S)-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene.
在存在或不存在环氧水解酶的情况下，使用由细胞色素P450 1A1、NADPH-细胞色素c还原酶和脂质组成的纯化重组系统，用底物7,8-二氢苯并[a]芘对细胞色素P450 1A1的活性位点进行了探究。发现底物的周转量为38 nmol/每nmol细胞色素P450/分钟。鉴定出的代谢产物有：酚类7,8-二氢苯并[a]芘（20 - 29%）；9,10-环氧-7,8,9,10-四氢苯并[a]芘（17 - 28%）；苯并[a]芘（12 - 19%）；7-羟基-7,8-二氢苯并[a]芘（13 - 16%）；8-羟基-7,8-二氢苯并[a]芘（7 - 15%）；3-羟基苯并[a]芘（7 - 15%）；4,5-环氧-4,5,7,8-四氢苯并[a]芘（0 - 4%）；以及7,8,9,10-四氢苯并[a]芘的一种三醇（0 - 4%）。9,10-环氧-7,8,9,10-四氢苯并[a]芘通过水在C-10处的苄基进攻快速水解为顺式和反式-9,10-二羟基-7,8,9,10-四氢苯并[a]芘（2:1）。大约71%的反式二醇衍生自(+)-(9S,10R)-环氧-7,8,9,10-四氢苯并[a]芘，这表明细胞色素P450 1A1对7,8-二氢苯并[a]芘对映异位面的选择性环氧化具有超过2:1的偏好。这种立体选择性与先前描述的细胞色素P450 1A1活性位点模型预测的假定立体选择性一致。纯形式或肝微粒体中的环氧水解酶催化9,10-环氧-7,8,9,10-四氢苯并[a]芘的水解，其受到1,1,1-三氯丙烷2,3-氧化物的抑制。环氧化物的(+)-(9S,10R)-异构体作为底物比其对映体略受青睐，并且通过苄基和非苄基进攻被裂解。对于(-)-(9R,10S)-9,10-环氧-7,8,9,10-四氢苯并[a]芘，仅发现苄基进攻。
