A serious challenge faced by an enzyme's active site is to avoid tight binding of the altered substrate in the ground state, as contrasted with the active site's very high affinity for the altered structure in the transition state. In the first part of this project, modified nucleotides, point mutations and structure determination will be used to investigate the binding properties of yeast orotidine 5'-phosphate decarboxylase, an unusually powerful catalyst. The principal goal of these experiments is to investigate the extent to which steric versus electrostatic interactions may be responsible for the instability of the ground state enzyme-substrate complex. In the second part of this project, structural methods, including mass spectrometry, will be used to investigate the extent to which the shortcomings of transition state analogues may arise from their possible tendency to trap water molecules at the active site, a possibility that is consistent with the observed thermodynamics of binding of transition states and transition state analogue inhibitors by E. coil cytidine deaminase. In the third part of this project, the rates of several uncatalyzed 2-substrate reactions, including aldol condensation, alcohol dehydrogenation, phosphorylation of alcohols by ATP, and peptidyl transfer, will be determined as a function of changing temperature. The goal of these experiments is to obtain benchmarks for comparison with rate constants to be obtained for the corresponding enzyme reactions, to estimate the affinity expected of an ideal bisubstrate analogue inhibitor of enzymes that catalyze each of these reactions, and to determine the extent to which the thermodynamics of activation and transition state binding by these enzymes may differ from those of enzymes catalyzing one-substrate and hydrolytic reactions. The hypotheses that will be tested in each of the three parts of this project have a significant bearing on the design of drugs and other enzyme inhibitors of practical interest.
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