Cytochrome b5 augments the 17,20-lyase activity of human P450c17 without direct electron transfer
Cytochrome b5 augments the 17,20-lyase activity of human P450c17 without direct electron transfer
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DOI：
10.1074/jbc.273.6.3158
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发表时间：
1998-02-06
影响因子：
4.8
通讯作者：
Miller, WL
中科院分区：
文献类型：
--
作者：
Auchus, RJ;Lee, TC;Miller, WL
在类固醇激素的生物合成中，P450 c17是催化α-碳类固醇的17 α-羟基化和裂解C-17，-C-20，-键以产生C-19，性类固醇的17，20-裂解酶活性的单一酶。细胞色素B（5）在体外增强细胞色素P450 c17的17，20-裂解酶活性，但这一点尚未在膜中得到证实，其作用机制尚不清楚。我们在酿酒酵母中表达了人P450 c17、人P450-氧化还原酶（OR）和/或人细胞色素B（5），并分析了所得酵母微粒体的17 α-羟化酶和17，20-裂解酶活性。仅表达P450 c17的酵母对δ（4）和δ（5）类固醇具有17 α-羟化酶和痕量17，20裂解酶活性。人OR与P450 c17的共表达使17 α-羟化酶和17，20-裂解酶反应的V-max增加了B倍;人B（5）与P450 c17的共表达也使17，20-裂解酶反应的V-max增加，但对17 α-羟化酶反应没有增加。将人B（5）与P450 c17和OR同时表达，或将纯化的人B（5）加入到来自共表达人P450 c17和OR的酵母的微粒体中，进一步增加了17，20-裂解酶反应的V-max，而不改变17 α-羟化酶活性。基因工程酵母和混合实验表明，OR是必要的和足够的微粒体17，20-裂解酶活性。将纯化的人holo-B（5）、apo-B（5）或细胞色素c加入含有人P450 c17和OR的微粒体中，证明B（5）的刺激作用不需要从B（5）向P450 c17的电子转移。这些数据表明，人B（5）主要作为变构效应物，主要与P450 c17 OR复合物相互作用，刺激17，20-裂解酶活性。
In the biosynthesis of steroid hormones, P450c17 is the single enzyme that catalyzes both the 17 alpha-hydroxylation of al-carbon steroids and the 17,20-lyase activity that cleaves the C-17,-C-20,, bond to produce C-19, sex steroids. Cytochrome b(5), augments the 17,20-lyase activity of cytochrome P450c17 in vitro, but this has not been demonstrated in membranes, and the mechanism of this action is unknown. We expressed human P450c17, human P450-oxidoreductase (OR), and/or human cytochrome b(5) in Saccharomyces cerevisiae and analyzed the 17 alpha-hydroxylase and 17,20-lyase activities of the resulting yeast microsomes. Yeast expressing only P450c17 have 17 alpha-hydroxylase and trace 17,20 lyase activities toward both Delta(4) and Delta(5) steroids. Coexpression of human OR with P450c17 increases the V-max,,, of both the 17 alpha-hydroxylase and 17,20-lyase reactions B-fold; coexpression of human b(5), with P450c17 also increases the V-max,, of the 17,20-lyase reactions but not of the 17 alpha-hydroxylase reactions. Simultaneous expression of human b(5), with P450c17 and OR, or addition of purified human b(5), to microsomes from yeast coexpressing human P450c17 and OR, further increases the V-max,, of the 17,20-lyase reaction without altering 17 alpha-hydroxylase activity. Genetically engineered yeast and mixing experiments demonstrate that OR is both necessary and sufficient for microsomal 17,20-lyase activity. Addition of purified human holo-b(5),, apo-b(5),, or cytochrome c to microsomes containing both human P450c17 and OR demonstrate that the stimulatory action of b(5), does not require electron transfer from b(5), to P450c17. These data suggest that human b(5), acts principally as an allosteric effector that interacts primarily with the P450c17 OR complex to stimulate 17,20-lyase activity.