Structural and functional analysis of NADPH-cytochrome P-450 reductase from human liver: complete sequence of human enzyme and NADPH-binding sites.
Structural and functional analysis of NADPH-cytochrome P-450 reductase from human liver: complete sequence of human enzyme and NADPH-binding sites.
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人肝脏 NADPH-细胞色素 P-450 还原酶的结构和功能分析：人酶和 NADPH 结合位点的完整序列。
DOI：
10.1021/bi00447a054
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发表时间：
1989
期刊：
影响因子：
2.9
通讯作者：
Shively,JE
中科院分区：
文献类型：
--
作者：
Haniu,M;McManus,ME;Birkett,DJ;Lee,TD;Shively,JE
摘要：用微序列分析和质谱法测定了人肝N-ADPH-细胞色素P-450还原酶的全氨基酸序列。总序列由676个氨基酸组成，由氨基末端乙酰基起始。没有翻译后修饰的证据，包括天冬酰胺连接的糖基化。该酶与其它哺乳动物酶的同源性为92-95%。序列差异主要集中在氨基端和羧基端的几个亲水区域。由于人酶与兔酶的免疫化学性质不同，尽管酶性质相似，但这些可变亲水区可能是潜在的抗原决定簇。NADPH耗尽的酶被异硫氰酸荧光素（赖氨酸特异性试剂）或5-（碘乙酰氨基）荧光素（半胱氨酸特异性试剂）灭活。在这两种情况下，NADP（H）的保护作用阻止了试剂对酶的失活。从5-（碘乙酰氨基）荧光素失活的酶中分离荧光肽，鉴定Cys 565为特异性NADPH保护的残基。e黄素蛋白NADPH-细胞色素P-450还原酶（EC 1.6. 2.4)是细胞色素P-450异生物质代谢系统的组成部分（威廉姆斯，1976;施瓦布和约翰逊，1987）。该酶系统在肝和肝外组织中均发现（McManus等人，1987 a; Hall等人，1989），并且定位于细胞的内质网和核膜中（Kasper，1971）。与细胞色素P-450一起，NADPH-细胞色素P-450还原酶对于在纯化系统中重建异生物质代谢活性是必需的。还原酶通过在催化过程中催化电子从NADPH转移到血红素蛋白而发挥作用（Lu & West，1978）。除了提供细胞色素P-450的还原等价物之外，还原酶还参与从NADPH到血红素加氧酶（Yoshida和菊池，1978）和细胞色素b5（Han等人，1981年）。该还原酶已从多种真核和原核组织中纯化（McManus等人，1989），包括人肝（Guengerich等，1981; Abraham等人，1986; McManus等人，1987 a，1989）和胎盘（Osawa等，1981; Muto & Tan，1986）。与由一个同工酶家族组成的细胞色素P-450相比，在所研究的每个物种中仅鉴定出一种形式的NADPH-细胞色素P-450还原酶，并且在小鼠中，
Revised Manuscript Received June 28, 1989 abstract: The complete amino acid sequence of human liver N ADPH-cy tochrome P-450 reductase has been determined by microsequence analysis and mass spectrometry. The total sequence consists of 676 amino acids initiated by an amino-terminal acetyl group. There is no evidence for posttranslational modifications, including Asn-linked glycosylation. The humanenzyme exhibits sequence homology in therange of 92-95% with other mammalian enzymes. Sequence differences were mainly confinedto several hydrophilic regions in the NH2-terminal and COOH-terminal domains. Since the human enzyme is immunochemically distinct from the rabbit enzyme despite similar enzymatic properties, it is likely that these variable hydrophilic regions are potential antigenic determinants. The NADPH-depleted enzyme is inactivated by either fluorescein isothiocyanate, a lysine-specific reagent, or 5-(iodoacetamido) fluorescein, a cysteine-specific reagent. In both cases, protection by NADP (H) prevents enzyme inactivation by thereagents. Isolation of fluorescent peptide from 5-(iodoacetamido) fluorescein-inactivated enzyme identified Cys 565 as the specifically NADPH-protected residue. e flavoprotein NADPH-cytochrome P-450 reductase (EC 1.6. 2.4) is an integral component of the cytochrome P-450 xenobiotic metabolizing system (Williams, 1976; Schwab & Johnson, 1987). This enzyme system is found in both hepatic and extrahepatic tissues (McManus et al., 1987a; Hall et al., 1989) and is localized in the endoplasmic reticulum and nu-clear membrane of cells (Kasper, 1971). Together with cy-tochrome P-450, N ADPH-cy tochrome P-450 reductase is essential for reconstitution of xenobiotic metabolizing activity in purified systems. The reductase functions by catalyzing electron transfer from NADPH to the hemoproteinduring catalysis (Lu & West, 1978). In additionto providing re-ducing equivalents to cytochrome P-450, the reductase is also involved in electron transferfrom NADPH to heme oxygenase (Yoshida & Kikuchi, 1978) and cytochrome b5 (Han et al., 1981). The reductase has been purified from a variety of eucaryotic and procaryotic tissues (McManus et al., 1989) including human liver (Guengerich et al., 1981; Abraham et al., 1986; McManus et al., 1987a, 1989) and placenta (Osawa et al., 1981; Muto & Tan, 1986). By contrast with cytochrome P-450, which is comprised of a family of isozymes, only one form of NADPH-cytochrome P-450 reductase has been identified in each species investigated, and in themouse, this