Zhang Haoming, Im Sang-Choul, Waskell Lucy
Department of Anesthesiology, University of Michigan Medical School, Ann Arbor, Michigan 48105, USA.
J Biol Chem. 2007 Oct 12;282(41):29766-76. doi: 10.1074/jbc.M703845200. Epub 2007 Aug 10.
The kinetics of product formation by cytochrome P450 2B4 were compared in the presence of cytochrome b(5) (cyt b(5)) and NADPH-cyt P450 reductase (CPR) under conditions in which cytochrome P450 (cyt P450) underwent a single catalytic cycle with two substrates, benzphetamine and cyclohexane. At a cyt P450:cyt b(5) molar ratio of 1:1 under single turnover conditions, cyt P450 2B4 catalyzes the oxidation of the substrates, benzphetamine and cyclohexane, with rate constants of 18 +/- 2 and 29 +/- 4.5 s(-1), respectively. Approximately 500 pmol of norbenzphetamine and 58 pmol of cyclohexanol were formed per nmol of cyt P450. In marked contrast, at a cyt P450:CPR molar ratio of 1:1, cyt P450 2B4 catalyzes the oxidation of benzphetamine congruent with100-fold (k = 0.15 +/- 0.05 s(-1)) and cyclohexane congruent with10-fold (k = 2.5 +/- 0.35 s(-1)) more slowly. Four hundred picomoles of norbenzphetamine and 21 pmol of cyclohexanol were formed per nmol of cyt P450. In the presence of equimolar concentrations of cyt P450, cyt b(5), and CPR, product formation is biphasic and occurs with fast and slow rate constants characteristic of catalysis by cyt b(5) and CPR. Increasing the concentration of cyt b(5) enhanced the amount of product formed by cyt b(5) while decreasing the amount of product generated by CPR. Under steady-state conditions at all cyt b(5):cyt P450 molar ratios examined, cyt b(5) inhibits the rate of NADPH consumption. Nevertheless, at low cyt b(5):cyt P450 molar ratios <or=1:1, the rate of metabolism of cyclohexane and benzphetamine is enhanced, whereas at higher cyt b(5):cyt P450 molar ratios, cyt b(5) progressively inhibits both NADPH consumption and the rate of metabolism. It is proposed that the ability of cyt b(5) to enhance substrate metabolism by cyt P450 is related to its ability to increase the rate of catalysis and that the inhibitory properties of cyt b(5) are because of its ability to occupy the reductase-binding site on cyt P450 2B4, thereby preventing reduction of ferric cyt P450 and initiation of the catalytic cycle. It is proposed that cyt b(5) and CPR compete for a binding site on cyt P450 2B4.
在细胞色素P450（cyt P450）与两种底物苄非他明和环己烷进行单催化循环的条件下，比较了细胞色素b5（cyt b5）和NADPH - cyt P450还原酶（CPR）存在时细胞色素P450 2B4形成产物的动力学。在单周转条件下，当cyt P450与cyt b5的摩尔比为1:1时，细胞色素P450 2B4催化底物苄非他明和环己烷的氧化，速率常数分别为18±2和29±4.5 s⁻¹。每nmol cyt P450大约形成500 pmol去甲苄非他明和58 pmol环己醇。形成鲜明对比的是，当cyt P450与CPR的摩尔比为1:1时，细胞色素P450 2B4催化苄非他明的氧化慢100倍（k = 0.15±0.05 s⁻¹），催化环己烷的氧化慢10倍（k = 2.5±0.35 s⁻¹）。每nmol cyt P450形成400 pmol去甲苄非他明和21 pmol环己醇。在等摩尔浓度的cyt P450、cyt b5和CPR存在时，产物形成是双相的，并且以cyt b5和CPR催化的快速和慢速速率常数发生。增加cyt b5的浓度会增加cyt b5形成的产物量，同时减少CPR产生的产物量。在所有检测的cyt b5与cyt P450摩尔比的稳态条件下，cyt b5抑制NADPH的消耗速率。然而，在低cyt b5与cyt P450摩尔比≤1:1时，环己烷和苄非他明的代谢速率会提高，而在较高的cyt b5与cyt P450摩尔比时，cyt b5会逐渐抑制NADPH的消耗和代谢速率。有人提出，cyt b5增强cyt P450底物代谢的能力与其提高催化速率的能力有关，并且cyt b5的抑制特性是由于其能够占据细胞色素P450 2B4上的还原酶结合位点，从而阻止铁细胞色素P450的还原和催化循环的启动。有人提出cyt b5和CPR竞争细胞色素P450 2B4上的一个结合位点。
