The objectives and specific aims of this research are to investigate the mechanism of action of aspartate aminotransferase (AATase) primarily through site-directed mutagenesis-and by chemical elaboration of cysteine residues introduced into the active site by the same technique. These mutant enzymes will be characterized physically by circular dichroism and differential scanning calorimetry in Berkeley; by crystallography in collaboration and by NMR in collaboration Kinetic and mechanistic characterization will be continued in Berkeley with rapid and conventional steady-state kinetics, fluorescence spectroscopy, and kinetic isotope effects as appropriate to each mutant. One mutant that has already been engineered to be a cationic amino acid- specific aminotransferase will be further developed in this direction by a rationally selected second-site mutation to increase the negative-charge density at the binding site, and by selecting arginine auxotrophs which have been transformed with the plasmid coding for the mutant AATase. It is possible that selective pressure will produce unanticipated second-site mutants. The health-related aspects of this work derive from the general role of pyridoxal phosphate-dependent enzymes in amino acid metabolism and of these enzymes in inborn errors of metabolism, such as homocystinuria, tyrosinemia, and valinemia. The physiological significant of the association of adjacent enzymes in metabolic pathways will be investigated by perturbing the contact areas by chemical modification and by site-directed mutagenesis. Observations on the stability of the complexes will be made by differential scanning calorimetry; and on the chemical properties of the associated AATase by fluorescence, circular dichroism, and by determining whether the product of the first enzyme is channeled directly to the active site of the second. The broader implications of the aspect of the research apply to the general phenomenon of the regulation of intermediary metabolism.
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