The aim of the research proposed is to better understand the mechanisms of the enzymes which are involved in carbohydrate metabolism; the storage and utilization of energy. The enzymes under study all use the substrate phosphoenolpyruvate (PEP) and require several cations for catalytic activity. the enzymes phosphoenolpyruvate carboxykinase (PEPCK), enolase and pyruvate kinase (PK) play key roles in gluconeogenesis and in glycolysis. The abnormal functioning of these enzymes are implicated in sudden infant death syndrome, secondary metabolic problems in diabetics, anemia and muscle functions. both PEPCK and PK are under metabolic control and catalyze homologous reactions. Each of these enzymes has a multication requirement, however, the roles of these cations have not ben described. Part of our research is directed toward the determination of the locations of the cation sites within the enzymes and in the elucidation of their catalytic and possibly regulatory functions. Structural information regarding these enzymes is scant, only PK has a crystal structure with reasonable resolution published. A portion of our research is directed toward unraveling the solution structures of the catalytic sites of these enzymes. The cation site on PEPCK will be located and the amino acid residues at the cation and substrate binding sites characterized. Binding of substrates will be refined. The role of the two cations binding to enolase will be unraveled. The amino acids responsible to substrate binding and catalysis will be determined by specific labeling and by NMR methods. Reaction rates and thermodynamic constants for the reaction at the catalytic site will be measured. Site-specific mutagenesis experiments will be initiated. Muscle and yeast PK will be investigated to understand the nature of allosteric regulation which occurs with the yeast enzyme. The location of the activator FDP and the structures of the substrates in the presence and absence of FDP will be determined. The structural changes at the active site upon activation will be studies by measuring metal- metal interactions. Site specific mutagenesis will be utilized to determine the roles of some of these residues. The development of new and more refined methods to help in our understanding of enzyme catalysis and regulation will be pursued. this information may help in modulating activity of these and analogous enzymes in vivo and in vitro.
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