STRUCTURAL STUDIES OF KETOSTEROID ISOMERASES

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 酮类固醇异构酶(KSI)是了解酶的作用机制和研究酶活性中心内残基定位功能基团催化作用的理想体系。该酶的突变体可以快速产生和纯化，并容易结晶。KSI催化类固醇底物中的碳-碳双键迁移，这是质子转移的结果。通过天冬氨酸碱基的初始去质子化生成双烯酸酯中间体，该中间体接受来自酪氨酸的氢键和氧阴离子空穴中质子化的天冬氨酸。现有的来自各种突变体的功能数据表明，KSI活性取决于活性位点附近保守残基相互作用所导致的碱基位置。该项目的目标是获得有或没有酶底物和中间体共结晶的影响酶活性的突变体的分子置换晶体结构，并确定催化活性差异的结构基础。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Ketosteroid isomerase (KSI) is an ideal system to understand enzyme mechanisms and investigate the catalytic contribution of positioning functional groups of residues within an enzyme active site. Mutants of the enzyme can be rapidly generated and purified and readily crystallized. KSI catalyzes the carbon-carbon double bond migration in a steroid substrate as the result of a proton transfer. Initial deprotonation by an aspartate base generates a dienolate intermediate, which accepts hydrogen bonds from a tyrosine and protonated aspartate in an oxyanion hole. Existing functional data from a variety of mutants suggest that the KSI activity depends on the positioning of the base by interactions of conserved residues adjacent to the active site. The goal of the project is to obtain molecular replacement crystal structures of mutants that affect the enzyme activity co crystallized with and without enzyme substrates and intermediates, and determine the structural basis for differences in the catalytic activity.