Lee-Robichaud P, Kaderbhai M A, Kaderbhai N, Wright J N, Akhtar M
Department of Biochemistry, University of Southampton, U.K.
Biochem J. 1997 Feb 1;321 ( Pt 3)(Pt 3):857-63. doi: 10.1042/bj3210857.
Human CYP17 (P-450(17alpha), 17alpha-hydroxylase-17,20-lyase)-catalysed side-chain cleavage of 17alpha-hydroxyprogestogens into androgens is greatly dependent on the presence of cytochrome b5. The native form of cytochrome b5 is composed of a globular core, residues 1-98, followed by a membrane insertable C-terminal tail, residues 99-133. In the present study the abilities of five different forms of cytochrome b5 to support the side-chain cleavage activity of CYP17 were compared. The five derivatives were: the native pig cytochrome b5 (native pig), its genetically engineered rat counterpart (core-tail), the soluble core form of the latter (core), the core with the secretory signal sequence of alkaline phosphatase appended to its N-terminal (signal-core) and the latter containing the C-terminal tail of the native rat protein (signal-core-tail). When examined by Edman degradation and MS, the engineered proteins were shown to have the expected N-terminal amino acid sequences and molecular masses. The native pig was found to be acetylated at the N-terminal. The native pig and core-tail enzymes were equally efficient at enhancing the side-chain cleavage activity of human CYP17 and the signal-core-tail was 55% as efficient. The core and signal-core constructs were completely inactive in the aforementioned reaction. All the five derivatives were reduced to varying degrees by NADPH:cytochrome P-450 (NADPH-P450) reductase and the relative efficiencies of this reduction were reminiscent of the behaviour of these derivatives in supporting the side-chain cleavage reaction. In the side-chain cleavage assay, however, NADPH-P450 reductase was used in large excess so that the reduction of cytochrome b5 derivatives was not rate-limiting. The results highlight that productive interaction between cytochrome b5 and CYP17 is governed not only by the presence of a membrane insertable hydrophobic region on the cytochrome b5 but also by its defined spatial orientation at the C-terminal.
人细胞色素P450 17α酶（CYP17，P - 450(17α)，17α - 羟化酶 - 17,20 - 裂解酶）催化17α - 羟基孕酮的侧链裂解生成雄激素，这在很大程度上依赖于细胞色素b5的存在。细胞色素b5的天然形式由一个球状核心（第1 - 98位氨基酸残基）和一个可插入膜的C末端尾巴（第99 - 133位氨基酸残基）组成。在本研究中，比较了五种不同形式的细胞色素b5支持CYP17侧链裂解活性的能力。这五种衍生物分别是：天然猪细胞色素b5（天然猪）、其基因工程改造的大鼠对应物（核心 - 尾巴）、后者的可溶性核心形式（核心）、在其N末端附加碱性磷酸酶分泌信号序列的核心（信号 - 核心）以及包含天然大鼠蛋白C末端尾巴的后者（信号 - 核心 - 尾巴）。通过埃德曼降解法和质谱分析，这些工程蛋白显示具有预期的N末端氨基酸序列和分子量。发现天然猪细胞色素b5在N末端被乙酰化。天然猪和核心 - 尾巴酶在增强人CYP17侧链裂解活性方面同样有效，而信号 - 核心 - 尾巴的效率为其55%。核心和信号 - 核心构建体在上述反应中完全无活性。所有这五种衍生物都被NADPH：细胞色素P450（NADPH - P450）还原酶不同程度地还原，这种还原的相对效率与这些衍生物在支持侧链裂解反应中的行为相似。然而，在侧链裂解测定中，NADPH - P450还原酶大量过量使用，因此细胞色素b5衍生物的还原不是限速步骤。结果突出表明，细胞色素b5与CYP17之间的有效相互作用不仅由细胞色素b5上可插入膜的疏水区域的存在决定，还由其在C末端的特定空间取向决定。
