Interaction of human CYP17 (P-450(17 alpha), 17 alpha-hydroxylase-17,20-lyase) with cytochrome b(5): Importance of the orientation of the hydrophobic domain of cytochrome b(5)

Human CYP17 (P-450(17 alpha), 17 alpha-hydroxylase-17,20-lyase)-catalysed side-chain cleavage of 17 alpha-hydroxyprogestogens into androgens is greatly dependent on the presence of cytochrome b(5). The native form of cytochrome b(5) 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 b(5) to support the side-chain cleavage activity of CYP17 were compared. The five derivatives were: the native pig cytochrome b(5) (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 b(5) derivatives was not rate-limiting. The results highlight that productive interaction between cytochrome b(5) and CYP17 is governed not only by the presence of a membrane insertable hydrophobic region on the cytochrome b(5) but also by its defined spatial orientation at the C-terminal.