STRUCTURAL STUDIES OF KETOSTEROID ISOMERASES

Stanford University, Stanford CA

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Abstract

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.