Long Term Objectives. Elucidate fundamental principles of catalytic efficiency, allosteric regulation and substrate channeling in enzyme catalysis.
Specific Aims. The work to be undertaken involves studies of the pyridoxal phosphate-requiring bacterial enzyme tryptophan synthase to accomplish the following: (a) Determine the mechanism of action of monovalent cations (MVC) in catalysis and regulation. (b) Establish a detailed mechanism for the roles of structural elements in the transmission of allosteric signals between subunits of the bienzyme complex. (c) Determine the catalytic function of protons thought to be involved in low-barrier H-bonds (LBHBs). Background. The tryptophan synthase bienzyme complex (alpha2-beta2) catalyzes the last two steps in the synthesis of L-Trp. Catalysis is intimately related to allosteric signaling and metabolite transfer between the alpha and beta sites. Reactions at the beta sites are hypothesized to switch alpha2-beta2 between low and high activity forms and """"""""open"""""""" and """"""""closed"""""""" conformations. This prevents the escape of the common metabolite, indole, and ensures that the alpha and beta reaction occur in phase. The binding of MVCs to a specific site (separate from the catalytic sites) activates specific steps in the catalytic pathway of tryptophan synthase. The MVC effect is hypothesized to result from selective lowering of the activation energies of certain steps through stabilizing interactions between the MVC and protein conformations complementary to the corresponding activated complexes. MVC binding also is essential to allosteric communication. Isotope effects suggest the involvement of an LBHB in tryptopha synthase catalysis. LBHBs are hypothesized to be strong bonds (12 to 24 kcal/mol) which lower activation energies, or stabilize conformation states. Methods. These hypotheses will be tested via the use of rapid kinetics, isotop effects, 1H NMR, mutants enzymes, and fluorescent probes. Significance to Huma Health and Disease. This work will contrbute new knowledge of structure-function relationships in enzyme catalysis and regulation concerning (a) The mechanism of action of MVCs. (b) The roles of conformation change in catalysis, allosteric regulation, and substrate channeling. (c) The roles, if any, of LBHBs in protein function.
