Pharmacogenetics Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
J Steroid Biochem Mol Biol. 1992 Dec;43(8):1031-6. doi: 10.1016/0960-0760(92)90330-L.
Mouse steroid 7α- and 15α-hydroxylases (P450c7 and P450c15) and coumarin 7-hydroxylase (P450coh) are structurally similar. To study the structural basis of the substrate specificities of these enzymes, we constructed a series of the mutant P450s, expressed in COS-1 and yeast cells, and studied them spectroscopically as well as enzyme-kinetically. A single amino acid mutation of residue-209 is sufficient to alter the substrate specificity of the P450s from xenobiotics to steroids and subsequently, from testosterone to corticosterone. Moreover, residue-209, when it is asparagine, appears to bind directly to the 11β-hydroxyl of corticosterone. The mutations also after the spin equilibrium of P450 depending on the hydrophobicity and size of residue-209. We conclude, therefore, that residue-209 resides close to the 6th ligand of heme in the mouse 2A subfamily and is located at a critical site of the substrate-binding pocket. As a result, the identity of the residue-209 plays a key role in determining the substrate specificity.
鼠甾体 7α-和 15α-羟化酶（P450c7 和 P450c15）和香豆素 7-羟化酶（P450coh）在结构上相似。为了研究这些酶的底物特异性的结构基础，我们构建了一系列突变 P450s，在 COS-1 和酵母细胞中表达，并从光谱学和酶动力学方面进行了研究。单个氨基酸残基 209 的突变足以改变 P450s 的底物特异性，从外源物质到类固醇，随后从睾酮到皮质酮。此外，当残基 209 为天冬酰胺时，它似乎直接结合到皮质酮的 11β-羟基上。这些突变还取决于残基 209 的疏水性和大小，改变了 P450 的自旋平衡。因此，我们得出结论，残基 209 靠近鼠 2A 亚家族血红素的第 6 个配体，位于底物结合口袋的关键部位。因此，残基 209 的身份在决定底物特异性方面起着关键作用。
