Purification, properties, and kinetic studies of cytoplasmic malate dehydrogenase from Taenia solium cysticerci.
Malate dehydrogenase (L: -malate: NAD oxidoreductase, EC 1.1.1.37) from the cytoplasm of Taenia solium cysticerci (cMDHTs) was purified 48-fold through a four-step procedure involving salt fractionation, ionic exchange, and dye affinity chromatography. cMDHTs had a native M (r) of 64,000, while the corresponding value per subunit, obtained under denaturing conditions, was 32,000. The enzyme is partially positive, with an isoelectric point of 8.7, and had a specific activity of 2,615 U mg(-1) in the reduction of oxaloacetate. The second to the 21st amino acids from cMDHTs N-terminal group were P G P L R V L I T G A A G Q I A Y N L S. This sequence is 100% identical to that of Echinococcus granulosus. Basic kinetic parameters were determined for this enzyme. The optimum pH for enzyme reaction was at 7.6 for oxaloacetate reduction and at 9.6 for malate oxidation. K (m) values for oxaloacetate, malate, NAD, and NADH were 2.4, 215, 50, and 48 microM, respectively. V (max) values for the substrates and cosubstrates as described above were 1,490, 87.8, 104, and 1,714 micromol min(-1) mg(-1). Several NAD analogs, structurally altered in either the pyridine or purine moiety, were observed to function as coenzymes in the reaction catalyzed by the purified malate dehydrogenase. cMDHTs activity was uncompetitive inhibited by arsenate for both the forward (Ki = 8.2 mM) and reverse (Ki = 77 mM) reactions. The mechanism of the cMDHTs reactivity was investigated kinetically by the product inhibition approach. The results of this study are qualitatively consistent with an Ordered Bi Bi reaction mechanism, in which only the coenzymes can react with the free enzyme.
Plancarte
Agustín
A
Departamento de Microbiología y Parasitología, Universidad Nacional Autónoma de México, UNAM, Mexico City, Mexico. apc@servidor.unam.mx
Nava
Gabriela
G
Mendoza-Hernández
Guillermo
G
D000818:Animals; D001149:Arsenates / Q000494:pharmacology; D005591:Chemical Fractionation; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003067:Coenzymes / Q000494:pharmacology; D003593:Cytoplasm / Q000201:enzymology*; D048209:Echinococcus granulosus / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015801:Helminth Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008293:Malates / Q000378:metabolism; D008970:Molecular Weight; D009243:NAD / Q000494:pharmacology; D062907:Oxaloacetic Acid / Q000378:metabolism; D021122:Protein Subunits; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D041201:Taenia solium / Q000201:enzymology*
D001149:Arsenates; D003067:Coenzymes; D004791:Enzyme Inhibitors; D015801:Helminth Proteins; D008293:Malates; D021122:Protein Subunits; D009243:NAD; D062907:Oxaloacetic Acid; C030298:malic acid; D008291:Malate Dehydrogenase; C025657:arsenic acid