ATOMIC RESOLUTION STUDIES OF KETOSTEROID ISOMERASE CATALYSIS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。酮类固醇异构酶(KSI)联盟项目是六个实验室(Boxer、Dawson、Herschlag、Kern、Petsko和Tonge)共同努力的成果，旨在应用多种互补方法，以前所未有的详细程度表征和理解酶催化作用。本项目的结晶学部分将侧重于获得与底物、中间类似物和产品结合的KSI上的原子分辨结构，以及含有非天然氨基酸的化学合成KSI的结构。这些结构研究的最终目标是(1)在原子分辨率下沿反应坐标的几个关键步骤研究野生型和突变型酶活性部位的结构变化；(2)使用各向异性位移参数精化和时间平均分子动力学精化来表征活性部位的原子涨落和构象异质性。这些信息将与热力学、动力学、光谱和计算结果结合使用，以严格表征

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The ketsosteroid isomerase (KSI) consortium project is a collaborative effort among six laboratories (Boxer, Dawson, Herschlag, Kern, Petsko, and Tonge) to apply multiple complementary approaches toward characterizing and understanding enzymatic catalysis in unprecedented detail. The crystallographic portion of this project will focus on obtaining atomic resolution structures on the KSI bound to substrate, an intermediate analog, and product, as well as the structures of chemically synthesized KSI containing non-natural amino acids. The ultimate goals of these structural studies are (1) to investigate, at atomic resolution, structural changes in the active sites of the wild-type and mutant enzymes at several key steps along the reaction coordinate and (2) to characterize atomic fluctuations and conformational heterogeneity in the active site using both anisotropic displacement parameter refinement and time averaged molecular dynamics refinement. This information will be used in combination with thermodynamic, kinetic, spectroscopic and computational results to rigorously characterize the physical basis of the catalytic power of th