Isotope partitioning for NAD-malic enzyme from Ascaris suum confirms a steady-state random kinetic mechanism.
Isotope partitioning studies beginning with E.[14C]NAD, E.[14C]malate, E.[14C]NAD.Mg2+, and E.Mg.[14C]malate suggest a steady-state random mechanism for the NAD-malic enzyme. Isotope trapping beginning with E.[14C]NAD and with varying concentrations of Mg2+ and malate in the chase solution indicates that Mg2+ is added in rapid equilibrium and must be added prior to malate for productive ternary complex formation. Equal percentage trapping from E.[14C]NAD.Mg and E.Mg.[14C]malate indicates the mechanism is steady-state random with equal off-rates for NAD and malate from E.NAD.Mg.malate. The off-rates for both do not change significantly in the ternary E.Mg.malate and E.NAD.Mg complexes, nor does the off-rate change for NAD from E.NAD. No trapping of malate was obtained from E.[14C]malate, suggesting that this complex is nonproductive. A quantitative analysis of the data allows an estimation of values for a number of the rate constants along the reaction pathway.
Chen
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CY
Department of Biochemistry, Texas College of Osteopathic Medicine/North Texas State University, Denton 76203.
Harris
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BG
Cook
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PF
D000818:Animals; D001200:Ascaris / Q000201:enzymology*; D002250:Carbon Radioisotopes; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000378:metabolism*; D008433:Mathematics; D008928:Mitochondria / Q000201:enzymology; D009243:NAD / Q000378:metabolism; D011865:Radioisotope Dilution Technique
D002250:Carbon Radioisotopes; D009243:NAD; D008291:Malate Dehydrogenase; C046446:malate dehydrogenase-(oxaloacetate-decarboxylating) (NAD+)