Human placental estrogen synthetase (aromatase). Effect of environment on the kinetics of protein-protein and substrate-protein interactions and the production of 19-oxygenated androgen intermediates in the purified reconstituted cytochrome P450 enzyme sy
Human placental estrogen synthetase (aromatase). Effect of environment on the kinetics of protein-protein and substrate-protein interactions and the production of 19-oxygenated androgen intermediates in the purified reconstituted cytochrome P450 enzyme sy
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人胎盘雌激素合成酶（芳香酶）。
DOI：
10.1016/0960-0760(91)90050-f
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发表时间：
1991
期刊：
影响因子：
--
通讯作者：
Bellino,FL
中科院分区：
文献类型：
--
作者：
Sethumadhavan,K;Bellino,FL
雌激素合成酶（芳香酶）通过 C19 处的两次羟基化和随后的 C19-10 裂合酶反应，催化雄激素转化为雌激素。我们在此报告使用高度纯化的芳香酶细胞色素 P450 单加氧酶系统进行重建研究的结果，该酶系统的两种蛋白质成分（细胞色素 P450 和 NADPH-细胞色素 P450 还原酶）均从人类足月胎盘微粒体中获得。通过改变其中一种成分（细胞色素 P450、NADPH-细胞色素 P450 还原酶或雄激素底物的量），而另两种成分在四种不同环境（磷脂、非离子去污剂、磷脂和非离子去污剂的混合物以及单独的缓冲液）中保持恒定，我们获得了支持以下结论的证据。与单独的缓冲液相比，重构的酶活性更高，并且蛋白质组分在去污剂和/或脂质环境中表现出低得多的表观 Km 值。尽管在大多数情况下，每种芳香酶蛋白组分的表观 Kman 和 Vmax 值因特定的限制组分和环境而显着不同，但催化效率 (Kcat/Km) 与限制蛋白组分无关，仅随环境变化（脂质-去污剂混合物中最高，单独脂质中最低）。当雄激素底物（雄烯二酮或睾酮）浓度在芳香酶蛋白成分（NADPH-细胞色素 P450 还原酶饱和）恒定量下变化时，雄烯二酮的 Km 较低，Vmax 较高。特异性常数 (Vmax/Km) 是重构环境的函数（单独的脂质中最高，单独的去垢剂中最低），并且平均而言，在特定环境中雄烯二酮大约高出 4 倍。使用雄烯二酮作为底物，在三种不同的疏水环境中，相对于 NADPH-细胞色素 P450 还原酶组分，在三种不同水平的芳香酶细胞色素 P450（亚饱和、饱和、过饱和）下检查 19-氧化雄激素中间体（19-羟基和 19-氧代雄烯二酮）的产生程度。两种 19-氧化雄激素，每种都以相对于对照相当的量制备，在去污剂-脂质混合环境中的细胞色素 P450 过饱和条件下可分离出最大量，而在仅脂质环境中的细胞色素 P450 过饱和条件下可分离出最小量。基于这些数据，我们提出，当细胞色素P450和NADPH-细胞色素P450还原酶形成瞬时复合物时，19-氧化雄激素中间体以逐步的方式顺序生物合成，并且可分离的19-氧化雄激素的量与过量细胞色素P450组分的量成正比。
Estrogen synthetase (aromatase) catalyzes the conversion of androgen into estrogen via two hydroxylations at C19and a subsequent C19-10lyase reaction. We report here the results of a reconstitution study using a highly purified aromatase cytochrome P450 monooxygenase enzyme system, with both protein components (cytochrome P450 and NADPH-cytochrome P450 reductase) obtained from human term placental microsomes. By varying one of the components (amounts of cytochrome P450, NADPH-cytochrome P450 reductase, or androgen substrate) as the other two were held constant in four different environments (phospholipid, non-ionic detergent, mixture of phospholipid and non-ionic detergent and buffer alone), we obtained evidence supporting the following conclusions. The reconstituted enzyme is more active and the protein components exhibit much lower apparent Kmvalues in the detergent and/or lipid environment compared with buffer alone. Although the apparent Kmand Vmaxvalues for each aromatase protein component differ significantly in most cases with the particular limiting component and environment, the catalytic efficiency (Kcat/Km) was independent of the limiting protein component and varied with the environment only (highest in the lipid-detergent mixture and lowest in lipid alone). When the concentration of androgen substrate (androstenedione or testosterone) was varied at constant amounts of the aromatase protein components (NADPH-cytochrome P450 reductase saturating), the Kmwas lower and the Vmaxwas higher for adrostenedione. The specificity constant (Vmax/Km) was a function of the reconstitution environment (highest in lipid alone and lowest in detergent alone) and was, on average, about 4-fold higher for androstenedione in a particular environment. The extent of production of 19-oxygenated androgen intermediates (19-hydroxy and 19-oxo androstenedione) was examined at three different levels of aromatase cytochrome P450 (subsaturating, saturating, super-saturating) relative to the NADPH-cytochrome P450 reductase component in the three different hydrophobic environments using androstenedione as substrate. Both 19-oxygenated androgens, each made in comparable amounts relative to control, were isolatable in greatest amounts under cytochrome P450 super-saturating conditions in the detergent-lipid mixed environment, and in least amounts under cytochrome P450 subsaturating conditions in the lipid-only environment. Based on these data, we propose that 19-oxygenated androgen intermediates are biosynthesized sequentially in a step-wise fashion as the cytochrome P450 and NADPH-cytochrome P450 reductase form transient complexes, and that the amount of isolatable 19-oxygenated androgen is proportional to the amount of excess cytochrome P450 component.