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.
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