P-enolpyruvate carboxykinase and pyruvate carboxylase catalyze the formation of oxaloacetate from three-carbon substrates (P-enolpyruvate or pyruvate), nucleotide triphosphates (GTP or ATP) and carbon dioxide or bicarbonate. Both reactions require one metal ion in the formation of the nucleotide metal complex and one or more additional metal ions to form catalytically competent complexes. Both enzymes are essential steps in the pathway of gluconeogenesis from lactate or alanine in mammals. The steady-state kinetic properties of these enzymes have been characterized, however many of the important features of catalysis are poorly understood. One purpose of these studies will be to characterize the interactions of substrates and metals at the catalytic sites of the enzymes. The interactions of substrates or substrate analogues with metal ions will be measured in electron paramagnetic resonance experiments which can establish direct metal substrate contacts and quantitate metal water and metal enzyme contacts. The rates of formation and dissociation of enzyme-substrate complexes will be established by substrate trapping experiments using labeled substrates. Reversibility of catalytic steps will be estimated by positional isotopic exchange experiments which can be quantitated by nuclear magnetic resonance or mass spectrometry. Heavy-atom kinetic isotope effects will be used to investigate the effects of allosteric activation on pyruvate carboxylase. These experiments will attempt to establish coordination of the nucleotide-bound metal and the enzyme-bound metals in the catalytic sites of both enzymes. Phosphobiotin or carboxyphosphate will be implicated as the intermediate in the pyruvate carboxylase reaction. The rates of catalytic steps and their reversibility will allow analysis of commitments to catalysis. These can then be used to establish the mechanism by which the allosteric activator, acetyl-CoA, activates pyruvate carboxylase and the metal activator, Mn(II), activates P-enolpyruvate carboxykinase. Heavy-atom kinetic isotope effects may be able to distinguish between changes in the transition state structure and changes in rate-limiting step(s) of pyruvate carboxylase in response to the allosteric activator.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
7R01GM036604-03
Application #
3290920
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1987-08-01
Project End
1991-03-31
Budget Start
1987-08-01
Budget End
1988-03-31
Support Year
3
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Albert Einstein College of Medicine
Department
Type
Schools of Medicine
DUNS #
009095365
City
Bronx
State
NY
Country
United States
Zip Code
10461
Werneburg, B G; Ash, D E (1997) VO2+(IV) complexes with pyruvate carboxylase: activation of oxaloacetate decarboxylation and EPR properties of enzyme-VO2+ complexes. Biochemistry 36:14392-402
Reczkowski, R S; Ash, D E (1994) Rat liver arginase: kinetic mechanism, alternate substrates, and inhibitors. Arch Biochem Biophys 312:31-7
Werneburg, B G; Ash, D E (1993) Chemical modifications of chicken liver pyruvate carboxylase: evidence for essential cysteine-lysine pairs and a reactive sulfhydryl group. Arch Biochem Biophys 303:214-21
Kanyo, Z F; Chen, C Y; Daghigh, F et al. (1992) Crystallization and oligomeric structure of rat liver arginase. J Mol Biol 224:1175-7
Chen, C Y; Sato, Y; Schramm, V L (1991) Isotope trapping and positional isotope exchange with rat and chicken liver phosphoenolpyruvate carboxykinases. Biochemistry 30:4143-51
Chen, C Y; Emig, F A; Schramm, V L et al. (1991) Inactivation of chicken mitochondrial phosphoenolpyruvate carboxykinase by o-phthalaldehyde. J Biol Chem 266:16645-52
Wente, S R; Villalba, M; Schramm, V L et al. (1990) Mn2(+)-binding properties of a recombinant protein-tyrosine kinase derived from the human insulin receptor. Proc Natl Acad Sci U S A 87:2805-9
Merkler, D J; Brenowitz, M; Schramm, V L (1990) The rate constant describing slow-onset inhibition of yeast AMP deaminase by coformycin analogues is independent of inhibitor structure. Biochemistry 29:8358-64
Merkler, D J; Schramm, V L (1990) Catalytic and regulatory site composition of yeast AMP deaminase by comparative binding and rate studies. Resolution of the cooperative mechanism. J Biol Chem 265:4420-6
Ash, D E; Emig, F A; Chowdhury, S A et al. (1990) Mammalian and avian liver phosphoenolpyruvate carboxykinase. Alternate substrates and inhibition by analogues of oxaloacetate. J Biol Chem 265:7377-84

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