STRUCTURAL STUDIES OF KETOSTEROID ISOMERASES

酮类固醇异构酶的结构研究

• 批准号: 8362048
• 负责人: ANA M GONZALEZ
• 金额: $ 0.36万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8362048
• 关键词: Active Sites; Affect; Aspartate; Carbon; Data; Enzymes; Funding; Goals; Grant; Hydrogen Bonding; Immigration; Isomerase; Ketosteroids; Molecular; National Center for Research Resources; Positioning Attribute; Principal Investigator; Protons; Radiation; Research; Research Infrastructure; Resources; Source; Steroids; Structure; System; Tyrosine; United States National Institutes of Health; base; cost; deprotonation; enzyme activity; enzyme mechanism; enzyme substrate; functional group; mutant; pi bond; structural biology

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.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 酮甾异构酶（KSI）是研究酶作用机理和研究酶活性中心功能基团定位的理想体系。 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。 天冬氨酸碱的初始去质子化产生二烯醇中间体，其在含氧阴离子空穴中接受来自酪氨酸和质子化天冬氨酸的氢键。 现有的各种突变体的功能数据表明，KSI活性取决于通过与活性位点相邻的保守残基的相互作用定位的碱基。该项目的目标是获得影响酶活性的突变体的分子置换晶体结构，这些突变体在有和没有酶底物和中间体的情况下共结晶，并确定催化活性差异的结构基础。