9729609 Cook The objective of this research is a determination of the mechanism of O-acetylserine sulfhydrylase (OASS-A), a pyridoxal phosphate (PLP) dependent enzyme . The kinetic and chemical mechanisms of OASS-A have been determined, and the three dimensional structure of the enzyme has been solved. A number of questions still remain concerning the mechanism. The slow steps in the first half of the reaction have been identified, but virtually nothing is known concerning the second half of the reaction. In this project, the reaction mechanism will be probed using presteady state and steady state kinetic techniques. Kinetic isotope effects will be used to obtain information on the second half of the reaction. The stereochemistry of the elimination of acetic acid and the addition of H2S, and whether a quinonoid intermediate exists along the reaction pathway will also be examined. Oligonucleotide-directed mutagenesis will be used to identify the enzyme residue that interacts with the acetyl side chain of OAS, groups that interact with the cofactor, and residue(s) involved in binding the alpha-carboxyl of OAS. The long term goal remains an elucidation of the mechanism of the multienzyme complex cysteine synthetase, composed of serine transacetylase and OASS. A complete description of the mechanisms of the component enzymes alone and in complex will be determined. The present project, however, focuses on the study of OASS-A. A study of the mechanism catalyzed by OASS-A should significantly increase our understanding of PLP enzymes in general, and those that catalyze beta-replacement reactions. Isotope effects is a technique that allows one to look at the structure of the activated complex (transition state) as reactant is converted to product, and the types of bonds formed and broken during the OASS reaction and the differences in chemistry compared to other PLP enzymes make OASS-A of interest. A knowledge of transition state structure provides a basis f or bactericides for PLP-dependent enzymes catalyzing beta-replacement reactions. The OASS-A reaction provides potential for the production of a number of novel beta-substituted amino acids.
