Kinetic mechanism of cytochrome P450 reductase from the house fly (Musca domestica)
Kinetic mechanism of cytochrome P450 reductase from the house fly (Musca domestica)
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DOI：
10.1016/s0965-1748(98)00131-3
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发表时间：
1999-03-01
影响因子：
3.8
通讯作者：
Feyereisen, R
中科院分区：
文献类型：
--
作者：
Murataliev, MB;Ariño, A;Feyereisen, R
用阴离子交换和亲和层析纯化了重组家蝇Ay细胞色素P450还原酶。细胞色素c还原酶活性的稳态动力学表明，以P450还原酶-NADPH-电子受体三元络合物为催化中间体，其反应机理为随机的BiBi型。NADP(H)结合是氢离子快速转移到FAD以及电子从FMN转移到细胞色素c所必需的。还原的细胞色素c对酶的活性没有影响，而NADP(+)和2‘-AMP对NADPH竞争性抑制P450还原酶，对细胞色素c非竞争性抑制P450还原酶。P450还原酶对NADPH的亲和力是对NADP(+)的10倍(K-d分别为0.31和3.3mU)。催化过程中亲和力的变化可能是FAD氧化还原电位+30 mV移动的原因。Cys560通过定点突变取代了Tyr。该突变通过降低核苷酸结合的双分子速率常数使酶对NADPH的亲和力降低35倍，对动力学机制没有可检测到的影响。与野生型相比，C560Y突变体酶对NADP(+)的亲和力降低了9倍，而对2‘-AMP的亲和力没有明显影响，这表明Cys560位于溶液中具有活性的全型酶的烟酰胺结合部位。(C)1999爱思唯尔科学有限公司。保留所有权利。
Recombinant house Ay (Musca domestica) cytochrome P450 reductase has been purified by anion exchange and affinity chromatography. Steady-state kinetics of cytochrome c reductase activity revealed a random Bi-Bi mechanism with formation of a ternary P450 reductase-NADPH-electron acceptor complex as catalytic intermediate. NADP(H) binding is essential for fast hydride ion transfer to FAD, as well as for electron transfer from FMN to cytochrome c. Reduced cytochrome c had no effect on the enzyme activity, while NADP(+) and 2'-AMP inhibited P450 reductase competitively with respect to NADPH and noncompetitively with respect to cytochrome c. The affinity of the P450 reductase to NADPH is 10 times higher than to NADP(+) (K-d of 0.31 and 3.3 mu M, respectively). Such an affinity change during catalysis could account for a +30 mV shift of the redox potential of FAD.Cys560 was substituted for Tyr by site-directed mutagenesis. This mutation decreased enzyme affinity to NADPH 35-fold by decreasing the bimolecular rate constant of nucleotide binding with no detectable effect on the kinetic mechanism. The affinity of the C560Y mutant enzyme to NADP(+) decreased 9-fold compared to the wild-type enzyme, while the affinity to 2'-AMP was not significantly affected, suggesting that Cys560 is located in the nicotinamide binding site of the active, full-size enzyme in solution. (C) 1999 Elsevier Science Ltd. All rights reserved.