VISUALIZING CATALYTIC INTERMEDIATES AND STRUCTURAL MECHANISM FOR ISOCITRATE DEHYDROGENASE USING STEADY-STATE AND SINGLE-TURNOVER LAUE EXPERIMENTS. Barry Stoddard, Basic Science & Stuctural Biology, Fred Hutchinson Cancer Research Center, Seattle WA, 98104

A combination of intermediate trapping methodologies and fast diffraction techniques are showing great promise for the direct visualization of structural intermediates formed during turnover. The greatest challenge for such studies is the matter of how the experimenter might induce a relatively high occupancy population throughout the crystal that represents a predominant catalytic species. This is of particular interest when enzymes are studied that normally follow multi-intermediate pathways with efficient free-energy profiles containing no substantial rate barriers between bound substrates and final products. In this talk, we present a series of studies of the enzyme isocitrate dehydrogenase, in which three specific catalytic states are trapped and visualized. The most important experimental lesson is that a combination of several techniques may be used to conduct such studies: kinetic analysis and spectroscopy in solution and the crystal, genetic mutational engineering, steady-state and single-turnover Laue experiments. When true fast Laue diffraction experiments are conducted using photochemical triggering, particular attention must be paid to substrate on-rates in the crystal lattice and mother liquor. In addition,independent experimental verification of structural assignments and of dynamic movements and interactions must be conducted: we present the recent use of molecular dynamic simulations and secondary mutagenic and kinetic studies to test specific features of the structural mechanism of IDH.