The pyruvate-proton exchange reaction of malic enzyme from pigeon liver.
Malic enzyme (L-malate:NADP+ oxidoreductase (decarboxylating) EC 1.1.1.40) catalyzes the incorporation of proton from medium water into pyruvate present either as the initial substrate or as the enzyme-bound product of malate decarboxylation. In the later reaction a single proton is incorporated into the methyl group of pyruvate. The pyruvate-medium proton exchange reaction requires Mg2+, NADPH and CO2-HCO3- as cofactors. The apparent Michaelis constants of pyruvate, NADPH and CO2-HCO3- are 4.8 mM, 2 microM and approx. 9 microM, respectively. The experimentally determined incorporation of 2.5 tritium atoms from tritiated water into pyruvate indicates that all three methyl protons of this compound are stereochemically equivalent in the exchange reaction. These results are consistent with the postulated kinetic mechanism for the malate reaction (Hsu, R.Y., Lardy, H.A. and Cleland, W.W. (1967) J. Biol. Chem. 242, 5315--5322), which predicts the formation of an enolpyruvate intermediate during the reaction. The rate of malic enzyme-catalyzed detritiation of beta-tritiated pyruvate is unaffected by modification of an essential protein thiol group with 5,5'-dithiobis(2-nitrobenzoic acid) or KCN. Moreover, the native- and thiol-modified enzymes also catalyze the detritiation of beta-tritiated bromopyruvate at slower rates.
D000345:Affinity Labels / Q000494:pharmacology; D000818:Animals; D010856:Columbidae; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008291:Malate Dehydrogenase / Q000378:metabolism*; D011190:Potassium Cyanide / Q000494:pharmacology; D011773:Pyruvates / Q000494:pharmacology; D019289:Pyruvic Acid; D013861:Thiocyanates / Q000494:pharmacology; D014316:Tritium
D000345:Affinity Labels; D011773:Pyruvates; D013861:Thiocyanates; D014316:Tritium; C017092:bromopyruvate; D019289:Pyruvic Acid; D004228:Dithionitrobenzoic Acid; D008291:Malate Dehydrogenase; D011190:Potassium Cyanide