Abstract
Haemophilus influenzae malate dehydrogenase ((S)-malate: NAD + oxidoreductase EC 1.1.1.37) was purified 109-fold with a 26% recovery through a four-step procedure involving salt fractionation, hydrophobic and dye affinity chromatography. The purified enzyme was demonstrated to be a dimer of M r 61 000. Initial velocity studies of all four substrates in the forward and reverse reactions indicated a sequential mechanism for the enzyme. Product and dead-end inhibition studies were consistent with an ordered bi-bi mechanism in which NAD is the first substrate bound to the enzyme and NADH, the second product released. Several analogs of NAD structurally altered in either the pyridine moiety were observed to function as coenzymes in the reaction catalyzed by the purified malate dehydrogenase. Alterations in the purine portion of the dinucleotides had a more pronounced effect on the kinetic parameters observed in malate oxidation. The enzyme was inactivated by incubation with diethylpyrocarbonate, whereas no inactivation was observed with sulfhydryl reagents.
Published Version
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