ATOMIC RESOLUTION STUDIES OF KETOSTEROID ISOMERASE CATALYSIS

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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