Interactions among P450 enzymes when combined in reconstituted systems: Formation of a 2B4-1A2 complex with a high affinity for NADPH-cytochrome P450 reductase
Interactions among P450 enzymes when combined in reconstituted systems: Formation of a 2B4-1A2 complex with a high affinity for NADPH-cytochrome P450 reductase
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DOI：
10.1021/bi980674a
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发表时间：
1998-09-15
期刊：
影响因子：
2.9
通讯作者：
Cawley, GF
中科院分区：
文献类型：
--
作者：
Backes, WL;Batie, CJ;Cawley, GF
本研究的目的是研究混合重组体系中P450 1A2、P450 2B4和P450还原酶之间的相互作用。此前，我们的实验室证明了在某些底物的存在下，1A2可以影响2B4的催化特性[Cawley等人。(1995)生物化学34,1244-1247]。本研究的目的是区分两种模型来解释这些相互作用：一种模型是底物增加一种P450酶对还原酶的亲和力，另一种模型是底物通过形成1A2-2B4复合体来增加一种P450对还原酶的亲和力。根据该模型，1A2-2B4的1A2部分与还原酶形成高亲和力的络合物。用二桂酰磷脂酰胆碱重组还原酶、1A2和2B4，用2B4-还原酶和1A2-还原酶二元体系以及不同2B4：1A2比例的三元体系考察了还原酶浓度对7-戊氧基间苯二酚-O-脱烷基化反应的影响。在还原酶过饱和时，三元体系的2B4依赖活性与二元体系相比有显著的抑制作用。这些结果与还原酶-1A2-2B4三元复合体的形成一致，其中还原酶与1A2特异结合。在较高的还原酶浓度下，1A2上的还原酶结合部位变得饱和，结果与还原酶与两种P450酶(还原酶-1A2-2B4还原酶)形成的四元复合体的形成一致。用1A2优先底物7-乙氧基间苯二酚进行的模拟实验表明，在混合重组体系中，7-乙氧基间苯二酚-O-脱乙基被刺激，证明高亲和力的2B4-1A2-还原酶复合体具有功能活性，而不仅仅是抑制复合体。
The purpose of this study is to characterize the interactions among P450 1A2, P450 2B4, and P450 reductase in mixed reconstituted systems. Previously, our laboratory demonstrated that in the presence of certain substrates, 1A2 can influence the catalytic characteristics of 2B4 [Cawley et al. (1995) Biochemistry 34, 1244-1247]. The goal of the current study is to distinguish between two models to explain these interactions: one model where substrate increases the affinity of one P450 enzyme for the reductase, and another model where substrate increases the affinity of one P450 for the reductase through the formation of a 1A2-2B4 complex. According to this model, the 1A2 moiety of 1A2-2B4 forms a high-affinity complex with reductase. Reductase, 1A2, and 2B4 were reconstituted with dilauroylphosphatidylcholine, and the effect of reductase concentration on 7-pentoxyresorufin-O-dealkylation was examined with 2B4-reductase and 1A2-reductase binary systems, and in ternary systems containing different 2B4:1A2 ratios. At subsaturating [reductase], there was a dramatic inhibition of the 2B4-dependent activity in the ternary system as compared with the binary systems. These results are consistent with the formation of a ternary (reductase-1A2-2B4) complex where the reductase is bound specifically to 1A2. At higher reductase concentrations where the reductase-binding sites on 1A2 become saturated, the results are consistent with the formation of a quaternary complex in which reductase binds to both P450 enzymes (reductase-1A2-2B4-reductase). Analogous experiments using the 1A2-preferred substrate 7-ethoxyresorufin showed a stimulation of 7-ethoxyresorufin-O-deethylation in the mixed reconstituted system, demonstrating that the high-affinity 2B4-1A2-reductase complex was functionally active and not merely an inhibitory complex.