This work will be carried out to further our understanding of the mechanism of O-acetylserine sulfhydrylase specifically and -replacement reactions catalyzed by PLP enzymes in general. Two specific aims: First, the acid-base chemistry catalyzed by enzyme will be elucidated. This aspect will make use of initial velocity studies to measure various values for inhibitors competitive with the substrates as a function of pH. In addition, spectral intermediates along the reaction pathway with a finite lifetime will be titrated to determine whether enzyme and/or cofactor pK values are perturbed as the reaction proceeds. Second, the location and amount of limitation of rate determining steps will be determined. These studies will make use of primary and secondary deuterium, solvent deuterium and multiple isotope effects and their pH dependence. The isotope effect data should allow pinpointing the location of rate determining steps and permit a mechanistic framework for interpretation of covalent catalysis. Finally, positional isotope exchange in which the ester carbonyl oxygen is labeled with 180 will be used to determine whether product release in the first half-reaction is partially or completely rate limiting. The broad range of experiments proposed here is likely to give considerably more detailed insight into the mode of action of this important enzyme then is available today, and the prospect that this information may later on be used in some intriguing studies on the catalytic consequences of protein-protein interaction gives the project an additional dimension. Dr. Cook is well qualified to carry out this work.
