Mechanism of the B6 Enzyme O-Acetylserine Sulfhydrylase

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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 enzyme will be elucidated. The 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 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 considerable more detailed insight to the mode of action of this important enzyme then is available today, and the prospect studies on the catalytic consequences of protein- protein interaction gives the project and additional dimension. Dr. Cook is well qualified to carry out this work.