Richardson T H, Johnson E F
Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, California 92037.
J Biol Chem. 1994 Sep 30;269(39):23937-43.
A Ser/Thr difference at position 364 underlies a phenotypic difference between naturally occurring variants of microsomal cytochrome P450 2C3 in their capacity to catalyze the 6 beta-hydroxylation of progesterone, as well as in their sensitivity to the inhibitor 16 alpha-methyl-progesterone. Position 364 of P450 2C3 maps to a substrate contacting domain suggested by models for mammalian P450 enzymes based on the structure of P450,101. In this study, Thr-364 of P450 2C3v, a progesterone 6 beta- and 16 alpha-hydroxylase, was replaced by Gly, Asp, Asn, Val, Leu, or Ile. The latter three amino acids did not alter the regiospecificity of P450 2C3v, whereas the Gly, Asp, and Asn substitutions each produced enzymes with properties that correspond closely to the Ser mutant that catalyzes 16 alpha-hydroxylation but not 6 beta-hydroxylation. The former are distinguished from the latter amino acids by their greater hydrophobicity and size. In contrast, the 16 alpha-hydroxylase activity could be greatly diminished by the introduction of an alanine replacement for Val-113. This mutation conferred progesterone 21- and 17 alpha-hydroxylase activity to P450 2C3v at the expense of 16 alpha-hydroxylase activity, leaving the 6 beta-hydroxylase activity largely unaffected. Compound mutants displayed the additive effects of the two mutations. These results are consistent with two distinct orientations for the binding of progesterone to P450 2C3v, resulting in 6 beta- and 16 alpha-hydroxylation, respectively. The binding of progesterone in these two orientations can be modulated relatively independently by modifications of the two key amino acid residues at 113 and 364.
微粒体细胞色素P450 2C3天然存在的变体在催化孕酮6β-羟基化的能力以及对抑制剂16α-甲基孕酮的敏感性方面存在表型差异，这一差异的基础是第364位的丝氨酸/苏氨酸差异。基于P450 101的结构，哺乳动物P450酶模型表明P450 2C3的第364位映射到一个底物接触结构域。在本研究中，P450 2C3v（一种孕酮6β-和16α-羟化酶）的苏氨酸-364被甘氨酸、天冬氨酸、天冬酰胺、缬氨酸、亮氨酸或异亮氨酸取代。后三种氨基酸没有改变P450 2C3v的区域特异性，而甘氨酸、天冬氨酸和天冬酰胺取代分别产生了具有与催化16α-羟基化但不催化6β-羟基化的丝氨酸突变体密切对应的特性的酶。前者与后者氨基酸的区别在于它们更大的疏水性和尺寸。相比之下，用丙氨酸取代缬氨酸-113可大大降低16α-羟化酶活性。这种突变赋予P450 2C3v孕酮21-和17α-羟化酶活性，但以16α-羟化酶活性为代价，而6β-羟化酶活性基本不受影响。复合突变体显示出两种突变的加性效应。这些结果与孕酮与P450 2C3v结合的两种不同取向一致，分别导致6β-和16α-羟基化。通过修饰113和364位的两个关键氨基酸残基，可以相对独立地调节孕酮在这两种取向中的结合。
