Oxidation kinetics of ethanol by human cytochrome P450 2E1 - Rate-limiting product release accounts for effects of isotopic hydrogen substitution and cytochrome b(5) on steady-state kinetics
Oxidation kinetics of ethanol by human cytochrome P450 2E1 - Rate-limiting product release accounts for effects of isotopic hydrogen substitution and cytochrome b(5) on steady-state kinetics
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DOI：
10.1074/jbc.272.47.29643
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发表时间：
1997-11-21
影响因子：
4.8
通讯作者：
Guengerich, FP
中科院分区：
文献类型：
--
作者：
Bell, LC;Guengerich, FP
已知许多细胞色素P450（P450）2 E1底物在大鼠肝微粒体中对K-m（K-D = K-D（m）/K-H（m））显示出类似于5的动力学氘同位素效应，但对k（猫）没有影响（例如N-亚硝基二甲胺、乙醇和CH 2Cl 2）。我们观察到重组人P450 2 E1催化乙醇氧化的K-D（m）值为3-5。用氧替代物氢过氧化枯烯代替NADPH和O-2得到类似的结果。铁P450 2 E1的还原是快速的（k > 1000 min（-1）），即使在没有底物的情况下。这些结果表明，K-m增加的基础是在催化循环的后期。确定乙醇氧化的本征同位素效应（（D）k）（竞争性）为3.8，表明C-H键断裂是同位素敏感的。稳态前的研究显示了产物形成的爆发（k = 410 min（-1）），爆发幅度对应于P450浓度。乙醇的氘代导致了3.2的同位素效应对爆发速率的影响。我们的结论是，产物释放在P450 2 E1将乙醇氧化为乙醛的过程中是限速的。稳态动力学可以通过以下范例来描述，其中k（cat）近似于产物释放速率，并且K-m是分母由C-H键断裂速率主导的表达式。
A number of cytochrome P450 (P450) 2E1 substrates are known to show kinetic deuterium isotope effects of similar to 5 on K-m (K-D = K-D(m)/K-H(m)), but not on k(cat), in rat liver microsomes (e.g. N-nitrosodimethylamine, ethanol, and CH2Cl2). We observed K-D(m) values of 3-5 for recombinant human P450 2E1-catalyzed ethanol oxidation. Replacing NADPH and O-2 with the oxygen surrogate cumene hydroperoxide yielded similar results. Ferric P450 2E1 reduction was fast (k > 1000 min(-1)) even in the absence of substrate. These results indicate that the basis for the increase in K-m is in the latter portion of the catalytic cycle, The intrinsic isotope effect ((D)k) for ethanol oxidation was determined (competitively) to be 3.8, indicating that C-H bond cleavage is isotopically sensitive. Presteady-state studies showed a burst of product formation (k = 410 min(-1)), with the burst amplitude corresponding to the P450 concentration, Deuteration of ethanol resulted in an isotope effect of 3.2 on the rate of the burst. We conclude that product release is rate-limiting in the oxidation of ethanol to acetaldehyde by P450 2E1. The steady-state kinetics can be described by a paradigm in which the k(cat) approximates the rate of product release, and K-m is an expression in which the denominator is dominated by the rate of C-H bond breaking.