W0339

Static and Time-Resolved Studies of Enzyme Reaction Coordinates: Molecular Odysseys in Four Dimensions. Andrew Mesecar*, Barry Stoddard^¶, Bernie Santarsiero*, Sonia Larsen*, Kiira Ratia*, Magdalini Vamvouka*, ^¥Zhong Ren, ^§Reinhard Pahl and ^§Vukica Srajer. *Center for Pharmaceutical Biotechnology, Univ. of Illinois, Chicago, ^¶Fred Hutchinson Cancer Research Center, ^¥Renz Research Inc., and the ^§Univ. of Chicago.

One of the remaining and great unsolved questions in chemistry and biology from the twentieth century is "How do enzymes accelerate the rates of chemical reactions over 10¹⁷-times faster than their corresponding uncatalyzed reactions, and how do they achieve such high degrees of substrate specificity? The central hypothesis of our laboratory is that enzymes achieve these remarkable properties, not by some unconventional "Enzymagical" property or theory, but by combining general physical-chemical and structural properties with dynamic processes. We are attempting to elucidate the contribution of these properties to the isocitrate dehydrogenase (IDH), trihydroxytoluene dioxygenase (THT-DO), and organophosphorous hydrolase (OPH) catalyzed reactions, by constructing three-dimensional, atomic resolution molecular movies of enzyme catalysis based on experimental data from both static and time-resolved x-ray crystal structures. Because of the complex nature of these multi-step enzyme reaction coordinates, we are integrating a variety of techniques from the fields of chemistry, biology, and physics including steady-state and time-resolved crystallography, enzyme kinetics and isotope effects, synthetic organic chemistry, computational chemistry, and molecular biology, to reach our goals. The focus of this presentation will be on our experimental and computational approaches to the construction of a preliminary molecular movie of the reaction coordinate of isocitrate dehydrogenase. Our most recent studies on IDH catalysis reveal large structural changes during the decarboxylation and enolization steps of the reaction that were previously unobserved. Preliminary work on the THT-DO and OPH enzyme systems, including substrate-caging strategies and polychromatic Laue x-ray diffraction studies, will also be presented. This work is supported in part by grants from the U.S. Department of Energy and the Office of Naval Research (N000140210956).