Kinetic mechanism of NAD:malic enzyme from Ascaris suum in the direction of reductive carboxylation.
Initial velocity studies in the absence and presence of product and dead-end inhibitors suggest a steady-state random mechanism for malic enzyme in the direction of reductive carboxylation of pyruvate. For this quadreactant enzymatic reaction (Mn2+ is a pseudoreactant), initial velocity patterns were obtained under conditions in which two substrates were maintained at saturating concentrations while one reactant was varied at several fixed concentrations of the other. Data from the resulting reciprocal plots, analyzed in terms of a bireactant mechanism, are consistent with a sequential mechanism with an obligatory order of addition of metal prior to pyruvate. NAD is competitive against NADH whether pyruvate and CO2 are maintained at low or high concentrations, whereas it is noncompetitive against pyruvate and CO2. Thio-NADH, alpha-ketobutyrate, and nitrite were used as dead-end analogs of NADH, pyruvate, and CO2, respectively. Thio-NADH is competitive against NADH, whereas it is noncompetitive against pyruvate and CO2, in accordance with a random mechanism. alpha-Ketobutyrate and nitrite gave noncompetitive inhibition against all substrates. The noncompetitive patterns observed for alpha-ketobutyrate versus pyruvate and nitrite versus CO2 suggest binding of the inhibitor to both the E.Mn.NADH and E.Mn.NAD complexes. Primary deuterium isotope effects are equal on all kinetic parameters, in agreement with the random mechanism, and suggest equal off-rates for NAD from E.Mn.NAD as well as pyruvate and NADH from E.Mn.NADH.pyruvate. Data are consistent with an overall symmetry in the malic enzyme reaction in the two reaction directions with a requirement for metal bound prior to pyruvate and malate.
Mallick
S
S
Department of Biochemistry and Molecular Biology, Texas College of Osteopathic Medicine, Fort Worth 76107.
Harris
B G
BG
Cook
P F
PF
D000818:Animals; D001200:Ascaris / Q000201:enzymology*; D001667:Binding, Competitive; D008291:Malate Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D010084:Oxidation-Reduction
D008291:Malate Dehydrogenase; C046446:malate dehydrogenase-(oxaloacetate-decarboxylating) (NAD+)