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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK017049-17
Application #
3225689
Study Section
Biophysics and Biophysical Chemistry B Study Section (BBCB)
Project Start
1976-09-01
Project End
1994-08-31
Budget Start
1989-09-01
Budget End
1990-08-31
Support Year
17
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Notre Dame
Department
Type
Schools of Arts and Sciences
DUNS #
824910376
City
Notre Dame
State
IN
Country
United States
Zip Code
46556
Holyoak, Todd; Nowak, Thomas (2004) pH Dependence of the reaction catalyzed by avian mitochondrial phosphoenolpyruvate carboxykinase. Biochemistry 43:7054-65
Susan-Resiga, Delia; Nowak, Thomas (2004) Proton donor in yeast pyruvate kinase: chemical and kinetic properties of the active site Thr 298 to Cys mutant. Biochemistry 43:15230-45
Susan-Resiga, Delia; Nowak, Thomas (2003) The proton transfer step catalyzed by yeast pyruvate kinase. J Biol Chem 278:12660-71
Susan-Resiga, Delia; Nowak, Thomas (2003) Monitoring active site alterations upon mutation of yeast pyruvate kinase using 205Tl+ NMR. J Biol Chem 278:40943-52
Krautwurst, Hans; Roschzttardtz, Hannetz; Bazaes, Sergio et al. (2002) Lysine 213 and histidine 233 participate in Mn(II) binding and catalysis in Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase. Biochemistry 41:12763-70
Rios, Sandra E; Nowak, Thomas (2002) Role of cysteine 306 in the catalytic mechanism of Ascaris suum phosphoenolpyruvate carboxykinase. Arch Biochem Biophys 404:25-37
Holyoak, T; Nowak, T (2001) Structural investigation of the binding of nucleotide to phosphoenolpyruvate carboxykinase by NMR. Biochemistry 40:11037-47
Bollenbach, T J; Nowak, T (2001) Thermodynamic linked-function analysis of Mg(2+)-activated yeast pyruvate kinase. Biochemistry 40:13088-96
Bollenbach, T J; Nowak, T (2001) Kinetic linked-function analysis of the multiligand interactions on Mg(2+)-activated yeast pyruvate kinase. Biochemistry 40:13097-106
Hlavaty, J J; Nowak, T (2000) Characterization of the second metal site on avian phosphoenolpyruvate carboxykinase. Biochemistry 39:1373-88

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