The research in this project will investigate the possibility that in two enzyme systems, lactate dehydrogenase and purine nucleoside phosphorylase, dynamic motions of the protein are central to the catalytic event. The overall hypothesis for this investigation is that through molecular dynamics, mixed quantum mechanical molecular mechanical, and quantum chemistry calculations we may identify such important motions, which we have called 'promoting vibrations'. Preliminary investigations show such periodic motion in the NAD cofactor in lactate dehydrogenase imposed by protein vibration on a subpicosecond timescale. Specific residues involved in generating the promoting vibration will be identified. The chemistry core of this Program Project will synthesize mutant proteins. Then experimental tests conducted in the Callender lab will be compared with theoretical predictions of changes in rate to evaluate the importance of the promoting protein motion in catalysis. Theoretical approaches will include the Quantum Kramers methodology developed in the PI's group for the calculation of rates in complex systems, spectral density computations for the analysis of molecular dynamics to locate promoting vibrations, and Chandler's transition path sampling to understand what atomic motions are part of the reaction coordinate in the target systems. Should experimental evidence suggest motions that contribute to catalysis more slowly than the currently available theoretically accessible timescales, these timescales will be used to guide further theoretical development. Purine nucleoside phosphorylase has been shown experimentally and in preliminary computations to facilitate explusion of the leaving group via motion of a three-atom oxygen stack that destabilizes bonding electrons at the transition state. This is a new type of promoting vibration - an electronic promoting vibration. Full QM/MM computations based on Schramm's transition state structure will investigate this concept. As in lactate dehydrogenase, we will propose mutants, that will experimentally test this hypothesis, and the Schramm lab will perform spectroscopic and direct measurments. Implementation of this program will allow the determination on an atomic level, how protein dynamics contributes in relatively rare events to cause catalysis.
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