Insights into the catalytic mechanism and active-site environment of Comamonas testosteroni delta 5-3-ketosteroid isomerase as revealed by site-directed mutagenesis of the catalytic base aspartate-38.
Insights into the catalytic mechanism and active-site environment of Comamonas testosteroni delta 5-3-ketosteroid isomerase as revealed by site-directed mutagenesis of the catalytic base aspartate-38.
复制标题
通过催化碱基天冬氨酸 38 的定点诱变揭示睾丸酮丛毛单胞菌 δ 5-3-酮类固醇异构酶的催化机制和活性位点环境。
DOI：
10.1021/bi00043a032
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发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Benisek,WF
中科院分区：
文献类型：
--
作者：
Holman,CM;Benisek,WF
摘要：睾丸酮丛毛单胞菌的5 -3-酮类固醇异构酶（KSI）通过形成酶结合的二烯醇（酯）中间体催化多种5（6）和5（10）类固醇的异构化。Asp-38与催化作用密切相关，显然是作为质子穿梭剂。在本文中的KSI突变体D38 E和D38 H的详细动力学特性的结果。两种突变体都保留了显著的活性，其中kcat和kc/Km值比D38 N突变体大103- 104倍。结果允许定性评估酶催化能力对催化碱基的定位和化学性质的敏感性。kcat 5”AND/kcat 5“10”EST的比率的接近一致性最容易通过第二化学步骤（中间体二烯醇（酯）的再酮化）不是显著速率决定的机理来解释。D38 E和D38 H突变体的速率常数的pH依赖性被发现与早期的提议一致，即在活性位点中存在一个尚未鉴定的滴定官能团，并表明残基38的静电环境是疏水的且带正电荷。
Revised Manuscript Received August 15, 1995® abstract: A5-3-Ketosteroid isomerase (KSI) of Comamonas testosteroni catalyzes the isomerization of a wide variety of 5 (6) and 5 (10) steroidsthrough the formation of an enzyme bound dienol (ate) intermediate. Asp-38 has been strongly implicated in catalysis, apparently serving as a proton shuttle. In this paper the results of a detailed kinetic characterization of the KSI mutants D38E and D38H are presented. Both mutants retain significant activity, with kcat and kcJKm values 103— 104 times greater than the D38N mutant. The results allow for a qualitative assessment of the sensitivity of the enzymes catalytic capability to the positioning and chemical nature of the catalytic base. The near identity of the ratios of kcat5" AND/kcat5’l0" EST is most easily explained by a mechanism in which the second chemical step, reketonization of the intermediate dienol (ate), is not significantly rate determining. The pH dependence of the rate constants for the D38E and D38H mutants is found to be consistent with earlier proposals that an as yet unidentified titrating functional groupis present in the active site and indicates that theelectrostatic environment of residue 38 is hydrophobic and positively charged.