This proposal describes experiments to probe the mechanism of catalysis of proton transfer from carbon by enzymes and by pyridoxal 5'-phosphate (PLP), and to rationalize the rate acceleration for enzyme-catalyzed proton transfer and decarboxylation reactions. Triosephosphate isomerase (TIM) utilizes the 14 kcal/mol intrinsic binding energy of the phosphodianion group of the substrate (R)-glyceraldehyde 3-phosphate in stabilization of the transition state for enzyme-catalyzed proton transfer. We propose that TIM also utilizes the binding energy of the potent allosteric activator phosphite dianion to drive a conformational change that sequesters the minimal substrate glycolaldehyde in an active site with an environment that is favorable for proton transfer from carbon. We plan to: (1) Examine the activation of TIM by exogenous phosphite dianion toward deprotonation of the generic simple carbon acid acetaldehyde, as a test of our hypothesis that the binding of phosphite dianion to TIM serves primarily to """"""""engineer"""""""" an active site with an environment that favors enolization. (2) Probe the role of closure of the critical """"""""mobile loop"""""""" of TIM in proton transfer from simple carbon acids. (3) Probe whether the conformational change induced by interactions of the phosphodianion group of orotidine 5'-phosphate (OMP) with OMP-decarboxylase are utilized in a similar manner to """"""""engineer"""""""" an active site that favors transition state stabilization for substrate decarboxylation. We also propose to characterize the activation of the alpha-amino protons of glycine and alanine by a simple pyridoxal 5'-phosphate analog, and to evaluate the importance of tunneling of the proton through the reaction barrier for nonenzymatic proton transfer at carbon. An understanding of these processes in water is essential to an evaluation of their role in enzymatic catalysis. Advances in the understanding of enzyme catalysis from such mechanistic studies on enzymes and nonenzymatic reactions may prove critical for drug design, to the understanding of metabolic pathways and diseases, and to the resolution of other health-related questions.
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