Yun C H, Miller G P, Guengerich F P
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
Biochemistry. 2001 Apr 10;40(14):4521-30. doi: 10.1021/bi002906n.
Human cytochrome P450 (P450) 1A2 is involved in the oxidation of many important drugs and carcinogens. The prototype substrate phenacetin is oxidized to an acetol as well as the O-dealkylation product [Yun, C.-H., Miller, G. P., and Guengerich, F. P. (2000) Biochemistry 39, 11319-11329]. In an effort to improve rates of catalysis of P450 1A2 enzymes, we considered a set of p-alkoxyacylanilide analogues of phenacetin and found that variations in the O-alkyl and N-acyl substituents altered the rates of the two oxidation reactions and the ratio of acetol/phenol products. Moving one methylene group of phenacetin from the O-alkyl group to the N-acyl moiety increased rates of both oxidations approximately 5-fold and improved the coupling efficiency (oxidation products formed/NADPH consumed) from 6% to 38%. Noncompetitive kinetic deuterium isotope effects of 2-3 were measured for all O-dealkylation reactions examined with wild-type P450 1A2 and the E225I mutant, which has 6-fold higher activity. A trend of decreasing kinetic deuterium isotope effect for E225I > wild-type > mutant D320A was observed for O-demethylation of p-methoxyacetanilide, which follows the trend for k(cat). The set of O-dealkylation and acetol formation results for wild-type P450 1A2 and the E225I mutant with several of the protiated and deuterated substrates were fit to a model developed for the basic catalytic cycle and a set of microscopic rate constants in which the only variable was the rate of product formation (substrate oxygenation, including hydrogen abstraction). In this model, k(cat) is considerably less than any of the microscopic rate constants and is affected by several individual rate constants, including the rate of formation of the oxygenating species, the rate of substrate oxidation by the oxygenating species, and the rates of generation of reduced oxygen species (H(2)O(2), H(2)O). This analysis of the effects of the individual rate constants provides a framework for consideration of other P450 reactions and rate-limiting steps.
人类细胞色素P450（P450）1A2参与多种重要药物和致癌物的氧化过程。原型底物非那西丁被氧化生成乙酰氧基苯以及O-脱烷基产物[Yun, C.-H., Miller, G. P., and Guengerich, F. P. (2000) Biochemistry 39, 11319 - 11329]。为了提高P450 1A2酶的催化速率，我们研究了一组非那西丁的对烷氧基酰苯胺类似物，发现O-烷基和N-酰基取代基的变化改变了两种氧化反应的速率以及乙酰氧基苯/苯酚产物的比例。将非那西丁的一个亚甲基从O-烷基转移到N-酰基部分，使两种氧化反应的速率都提高了约5倍，并将偶联效率（形成的氧化产物/消耗的NADPH）从6%提高到38%。在用野生型P450 1A2和活性高6倍的E225I突变体进行的所有O-脱烷基反应中，测得的非竞争性动力学氘同位素效应为2 - 3。对于对甲氧基乙酰苯胺的O-去甲基化反应，观察到E225I > 野生型 > 突变体D320A的动力学氘同位素效应呈下降趋势，这与k(cat)的趋势一致。野生型P450 1A2和E225I突变体与几种质子化和氘代底物的O-脱烷基化及乙酰氧基苯形成结果符合为基本催化循环和一组微观速率常数建立的模型，其中唯一的变量是产物形成速率（底物氧化，包括氢提取）。在该模型中，k(cat)远小于任何微观速率常数，并受几个单独的速率常数影响，包括氧合物种的形成速率、氧合物种对底物的氧化速率以及还原氧物种（H(2)O(2)、H(2)O）的生成速率。对各个速率常数影响的这种分析为考虑其他P450反应和限速步骤提供了一个框架。
