Purification and kinetic characterization of Haemophilus parasuis malate dehydrogenase.
Haemophilus parasuis malate dehydrogenase ((S)-malate:NAD+ oxidoreductase; EC 1.1.1.37) isolated from cell sonicates was purified 584-fold to electrophoretic homogeneity with a 19% recovery and a specific activity of 222 units/mg protein. SDS-polyacrylamide gel electrophoresis and molecular exclusion chromatography indicated the purified enzyme to be a dimer composed of 34,600 molecular weight subunits. Kinetic parameters 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 mechanism in which NAD is the first substrate bound to the enzyme and NADH the second product released. Protection against thermodenaturation of the enzyme by NAD and not by malate was supportive of this mechanism. A pronounced product inhibition by NADH (K(i) = 9.0 microM) was observed. Although NADP did not serve as a coenzyme, a number of analogs of NAD structurally altered in the nitrogen base moieties were observed to function as coenzymes in the oxidation of malate catalyzed by the purified malate dehydrogenase. Coenzyme-competitive inhibition of the malate 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) = 18 microM). Although inhibition of growth of H. parasuis by this analog was observed, it was less effective (K(i) = 136 microM) than the inhibition of the purified dehydrogenase.
Wise
D J
DJ
Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg 24061-0308, USA.
Anderson
C D
CD
Anderson
B M
BM
D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D002850:Chromatography, Gel; D003067:Coenzymes / Q000378:metabolism / Q000494:pharmacology; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006190:Haemophilus / Q000201:enzymology* / Q000254:growth & development; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism*; D008293:Malates / Q000378:metabolism; D008970:Molecular Weight; D009243:NAD / Q000031:analogs & derivatives / Q000494:pharmacology; D009540:Nicotinyl Alcohol / Q000031:analogs & derivatives / Q000494:pharmacology; D011489:Protein Denaturation; D013379:Substrate Specificity; D013696:Temperature
C107321:3-pyridylcarbinol adenine dinucleotide; D003067:Coenzymes; D004791:Enzyme Inhibitors; D008293:Malates; D009243:NAD; C002098:3-aminopyridine adenine dinucleotide; D000244:Adenosine Diphosphate; C030298:malic acid; D009540:Nicotinyl Alcohol; D008291:Malate Dehydrogenase; D000241:Adenosine