The overall objective of this proposal is an understanding of enzyme mechanism. Efforts are concentrated on three enzyme systems including the malic enzyme, phospho-fructokinase, and aspartase. A three step chemical mechanism has been proposed for malic enzyme in which malate is first oxidized to oxalaceate, the oxalaceate intermediate is decarboxylated to enolpyruvate, and the latter is then tautomerized to pyruvate. Recent evidence obtained with alternative dinucleotide substrates suggests one of two possibilities. First, there is a change in the mechanism of the oxidative decarboxylation of malate to enolpyruvate from two steps to a concerted mechanism. Second, a secondary (13)C isotope effect is present during the oxidation of malate to the oxalacetate intermediate. The partitioning of the oxalacetate intermediate in the E:NADH:Mg:oxalacetate complex, toward malate and pyruvate will be used to probe this possible mechanism change. These studies will be carried out with protium and deuterium labeled reduced alternative dinucleotides and Mg(2+), Mn(2+) and Cd(2+). These studies will be followed up with secondary deuterium isotope effects using NAD-4-D and L-malate-3, 3-t2 to define the transition state structure for hydride transfer and decarboxylation (if present) steps. In addition, primary deuterium and tritium isotope effects will be used to study the reductive carboxylation reaction as well as the role of the metal ion. Studies will be extended to include the closely related isocitrate and delta-phosphoglucamate dehydrogenase to determine whether the phenomenon described above for malic enzymes is common to this class of oxidative decarboxylases. Preliminary evidence has been obtained to implicate a second metal ion (in addition to MgPPi) in the pyrophosphate phosphofructokinase (PPi-PFK) reaction. Exchange inert metal-PPi complexes will be used to test this hypothesis. The phosphoryl transfer step is rate determining for the PPi-PFK reaction and thus primary and secondary (18)O effects will be carried out using the remote label technique to probe transition state structure. The availability of a form of the ATP-PFK desensitized to hysteresis in the time courses for F6P phosphorylation and homotropic cooperativity has facilitated studies of the kinetic mechanism of regulation and the mechanism of acid-base catalysis. Initial velocity studies will be used to determine the effect of allosteric modulators along the reaction pathway, the mechanism of acid-base, catalysis, and the optimum protonation state for binding groups on reactants and effectors as well as the active and allosteric sites on enzyme. An E1cb mechanism has been proposed for aspartase in which C-N bond cleavage is rate determining. This proposed mechanism will be tested using protium washout in the NMR. In addition, the acid-base catalytic mechanism of the enzyme and optimum protonation state of binding groups will be determined.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM036799-08
Application #
3291258
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1985-09-19
Project End
1995-08-31
Budget Start
1991-09-01
Budget End
1992-08-31
Support Year
8
Fiscal Year
1991
Total Cost
Indirect Cost
Name
University of North Texas
Department
Type
Schools of Osteopathy
DUNS #
110091808
City
Fort Worth
State
TX
Country
United States
Zip Code
76107
Stanley, T M; Johnson Jr, W H; Burks, E A et al. (2000) Expression and stereochemical and isotope effect studies of active 4-oxalocrotonate decarboxylase. Biochemistry 39:718-26
Karsten, W E; Hwang, C C; Cook, P F (1999) Alpha-secondary tritium kinetic isotope effects indicate hydrogen tunneling and coupled motion occur in the oxidation of L-malate by NAD-malic enzyme. Biochemistry 38:4398-402
Yuen, M H; Mizuguchi, H; Lee, Y H et al. (1999) Crystal structure of the H256A mutant of rat testis fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase. Fructose 6-phosphate in the active site leads to mechanisms for both mutant and wild type bisphosphatase activities. J Biol Chem 274:2176-84
Karsten, W E; Chooback, L; Liu, D et al. (1999) Mapping the active site topography of the NAD-malic enzyme via alanine-scanning site-directed mutagenesis. Biochemistry 38:10527-32
Jagannatha Rao, G S; Cook, P F; Harris, B G (1999) Kinetic characterization of a T-state of Ascaris suum phosphofructokinase with heterotropic negative cooperativity by ATP eliminated. Arch Biochem Biophys 365:335-43
Mizuguchi, H; Cook, P F; Tai, C H et al. (1999) Reaction mechanism of fructose-2,6-bisphosphatase. A mutation of nucleophilic catalyst, histidine 256, induces an alteration in the reaction pathway. J Biol Chem 274:2166-75
Rosenbaum, K; Jahnke, K; Schnackerz, K D et al. (1998) Secondary tritium and solvent deuterium isotope effects as a probe of the reaction catalyzed by porcine recombinant dihydropyrimidine dehydrogenase. Biochemistry 37:9156-9
Cook, P F (1998) Mechanism from isotope effects. Isotopes Environ Health Stud 34:3-17
Karsten, W E (1997) Dihydrodipicolinate synthase from Escherichia coli: pH dependent changes in the kinetic mechanism and kinetic mechanism of allosteric inhibition by L-lysine. Biochemistry 36:1730-9
Chooback, L; Karsten, W E; Kulkarni, G et al. (1997) Expression, purification, and characterization of the recombinant NAD-malic enzyme from Ascaris suum. Protein Expr Purif 10:51-4

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