Conformational heterogeneity of cytochrome P450 3A4 revealed by high pressure spectroscopy
Conformational heterogeneity of cytochrome P450 3A4 revealed by high pressure spectroscopy
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DOI：
10.1016/j.bbrc.2003.09.247
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发表时间：
2003-12-05
影响因子：
3.1
通讯作者：
Hoa, GHB
中科院分区：
文献类型：
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作者：
Davydov, DR;Halpert, JR;Hoa, GHB
应用流体静压微扰法研究了溴隐亭（BCT）作为底物诱导的人细胞色素P450 3A4（CYP3A4）的跃迁。将纯化的酶在溶液中的正压行为与共表达CYP3A4、细胞色素B（5）、（B（5））和NADPH-细胞色素P450还原酶（CPR）的酿酒酵母重组微粒体中观察到的正压行为进行比较。在这两种情况下，检测到重要的正压异质性的CYP3A4。溶液中只有约70%的CYP3A4和约50%的微粒体酶对压力诱导的P450-> P420转变敏感。结果表明，无论是在溶液中和在膜CYP3A4是由两个构象具有不同的自旋平衡位置和不同的正压性质。在实验的时间范围内没有观察到这些构象之间的相互转化。重要的是，压力诱导的自旋位移，这是迄今为止研究的所有细胞色素P450的特征，仅在溶液中的CYP3A4中检测到; P450-> P420转换是在微粒体中检测到的唯一压力诱导过程。这一事实表明CYP3A4的高自旋状态的不寻常稳定性，这被认为反映了血红素蛋白与蛋白质伴侣（B（5）和CPR）和/或膜脂质的特异性相互作用导致血红素部分的水可及性降低。(C)2003年爱思唯尔公司All rights reserved.
We applied hydrostatic pressure perturbation to study substrate-induced transitions in human cytochrome P450 3A4 (CYP3A4) with bromocriptine (BCT) as a substrate. The barotropic behavior of the purified enzyme in solution was compared with that observed in recombinant microsomes of Saccharomyces cerevisiae coexpressing CYP3A4, cytochrome b(5), (b(5)) and NADPH-cytochrome P450 reductase (CPR). Important barotropic heterogeneity of CYP3A4 was detected in both cases. Only about 70% of CYP3A4 in solution and about 50% of the microsomal enzyme were susceptible to a pressure-induced P450-->P420 transition. The results suggest that both in solution and in the membrane CYP3A4 is represented by two conformers with different positions of spin equilibrium and different barotropic properties. No interconversion between these conformers was observed within the time frame of the experiment. Importantly, a pressure-induced spin shift, which is characteristic of all cytochromes P450 studied to date, was detected in CYP3A4 in solution only; the P450-->P420 transition was the sole pressure-induced process detected in microsomes. This fact suggests unusual stabilization of the high-spin state of CYP3A4, which is assumed to reflect decreased water accessibility of the heme moiety due to specific interactions of the hemoprotein with the protein partners (b(5) and CPR) and/or membrane lipids. (C) 2003 Elsevier Inc. All rights reserved.