NSF Award
9405020
9405020 Cook O-Acetylserine sulfhydrylase (OASS) is a PLP-dependent enzyme that catalyzes the last step in the biosynthesis of L-cysteine, the conversion of O-acetyl-L-serine and sulfide to L-cysteine and acetate. he OASS is one of only a few -substitution and - elimination reactions catalyzed by PLP-dependent enzymes that has been studied in detail. It is unique as a -substitution reaction in having a ping pong mechanism, allowing study of the two halves of the reaction separately. Over the past grant period studies have focused on the kinetic and acid-base chemical mechanisms of the A and B isozymes of OASS, as well as the spectral identification of intermediates, crystallization, and site-directed metagenesis. As a result, the kinetic and acid-base chemical mechanisms are well known, as is the spectral properties of many of the intermediates along the reaction pathway. In addition, OASS-A has been crystallized, apoenzyme prepared, and several site- directed mutants have been studied. Continuation of the studies of OASS will be carried out using four main aims. 1) Although a mechanism has been proposed for OASS, it is not known what intermediates build up along the reaction pathway, or the nature of the rate-determining transition state structures. Primary substrate deuterium solvent deuterium, secondary deuterium, and multiple isotope effects will be measured for both isozymes using natural and slow alternative substrates. 2) The isotope effect studies will be complemented by rapid scanning stopped-flow studies of both half reactions. Aims 1 and 2 should allow a reconstitution of the reaction pathway and provide transition state structural information. 3) A number of site-directed mutants have ben made probing the function of enzymes residues that are implicated in aspects of mechanism including K42 (Schiff base lysine), C43 (the only cysteine and known to be in the active site), and W51/W162 (one of the only two Trps is implicated fluorescence e nergy transfer) and these are being characterized. Additional site- directed mutants will be prepared as dictated by the enzyme crystal structure, which will be solved in addition to the studies outlines above, but is not part of the present proposal. the completion of these studies will be placed OASS among the best studies of the PLP enzymes. In addition, the structure of OASS-A will be available to compare to the tryptophan synthase 2 which catalyzes a very similar reaction. %%% O-Acetylserine sulfhydrylase is an eznyme that depends on a form of vitamin B6 for its activity. The purpose of the proposed studies is to determined how the enzyme and its B6 cofactor catalyze the substitution of one group for another in amino acids, the building block of proteins. The enzyme studies is representative of a class of enzymes, and thus anything learned could be applied to other enzymes in the class. The reaction will be studied using kinetic techniques which view the enzyme in the act of catalysis, and site directed mutagenesis which allows a change in specific amino acid residues of the enzyme thought to be important in the reaction. ***
