Kinetic mechanism in the direction of oxidative decarboxylation for NAD-malic enzyme from Ascaris suum.
Measurement of the initial rate of the malic enzyme reaction varying the concentration of NAD at several different fixed levels of Mg2+ (0.25-1.0 mM) and a single malate concentration gave a pattern which intersects to the left of the ordinate. Repetition of this initial velocity pattern at several additional malate concentrations and treatment in terms of a terreactant mechanism suggests an ordered mechanism in which NAD adds prior to Mg2+ which must add prior to malate. On the other hand, when a broader concentration range of Mg2+ (0.25-50 mM) is used, data are consistent with a random mechanism in which Mg2+ must add prior to malate. By use of product inhibition studies, pyruvate is competitive vs. malate and noncompetitive vs. NAD, while NADH is competitive vs. NAD and noncompetitive vs. malate. These results are consistent with the random addition of substrates and further suggest rapid equilibrium random release of products. Tartronate, a dead-end analogue of malate, is competitive vs. malate and noncompetitive vs. NAD. Thio-NAD is a slow substrate which is used at 2.4% the maximum rate of NAD. When used as a dead-end analogue of NAD, thio-NAD is competitive vs. NAD and gives a complex inhibition pattern vs. malate in which competitive inhibition is apparent at low concentrations of malate (less than 12.5 mM), and this changes to uncompetitive inhibition at high concentrations of malate (greater than 12.5 mM). These data are consistent with a steady-state random mechanism in the direction of oxidative decarboxylation in which Mg2+ adds in rapid equilibrium prior to malate.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D001200:Ascaris / Q000201:enzymology*; D001667:Binding, Competitive; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008291:Malate Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008293:Malates / Q000378:metabolism; D009243:NAD / Q000378:metabolism*; D011773:Pyruvates / Q000378:metabolism; D019289:Pyruvic Acid; D013646:Tartronates / Q000378:metabolism
D008293:Malates; D011773:Pyruvates; D013646:Tartronates; D009243:NAD; C032973:tartronic acid; C030298:malic acid; D019289:Pyruvic Acid; D008291:Malate Dehydrogenase; D008274:Magnesium