PURIFICATION AND KINETIC CHARACTERIZATION OF HAEMOPHILUS-PARASUIS MALATE-DEHYDROGENASE

Haemophilus parasuis malate dehydrogenase ((S)-malate:NAD(+) oxidoredu ctase; EC 1.1.1.37) isolated from cell sonicates was purified 584-fold to electrophoretic homogeneity with a 19% recovery and a specific act ivity of 222 units/mg proteins. SDS-polyacrylamide gel electrophoresis and molecular exclusion chromatography indicated the purified enzyme to be a dimer composed of 34,600 molecular weight subunits. Kinetic pa rameters for all four substrates in the forward and reverse reactions indicated a sequential mechanism for this enzymic process. Product and dead-end-inhibition studies were consistent with an ordered bi-bi mec hanism in which NAD is the first substrate bound to the enzyme and NAD H the second product released. Protection against thermodenaturation o f the enzyme by NAD and not by malate was supportive of this mechanism . A pronounced product inhibition by NADH (K-i = 9.0 mu M) was observe d. Although NADP did not serve as a coenzyme, a number of analogs of N AD structurally altered in the nitrogen base moieties were observed to function as coenzymes in the oxidation of malate catalyzed by the pur ified malate dehydrogenase. Coenzyme-competitive inhibition of the mal ate dehydrogenase was observed with five adenosine derivatives and six structural analogs of NAD. Of the NAD analogs studied as inhibitors, 3-pyridylcarbinol adenine dinucleotide was the most effective (K-i = 1 8 mu M). Although inhibition of growth of H. parasuis by this analog w as observed, it was less effective (K-i = 136 mu M) than the inhibitio n of the purified dehydrogenase. (C) 1997 Academic Press.