title: L-Malate dehydrogenase from Pseudomonas stutzeri: purification and characterization.
abstract: L-Malate dehydrogenase (MDH) from Pseudomonas stutzeri was purified to homogeneity by a two-step procedure comprising anion-exchange chromatography and affinity chromatography on immobilized anthraquinone alpha-ketocarboxyl biomimetic dye. The enzyme has molecular mass of 66,500 Da and consists of two identical subunits of molecular mass of approximately 34,000 Da. Initial velocity, product inhibition, and binding studies were consistent with an ordered Bi-Bi mechanism for the enzyme action and the formation of a ternary complex. The enzyme is susceptible to activation and inhibition by its substrates. Thermodynamic analysis and kinetic inhibition studies were performed for determining basic equilibrium and kinetic constants. Malate dehydrogenase was covalently inactivated by a dichlorotriazine dye, Vilmafix Blue A-R (VBAR). The inactivation process follows first-order kinetics, and the results from kinetic analysis suggested the formation of a noncovalent enzyme-dye complex prior to the covalent reaction, with Kd 84.6 microM and a maximum rate constant 0.16 min(-1). The enzyme inactivation process was partially inhibited by substrates and inhibitors. Quantitatively inactivated MDH contained approximately 1 mole of dye per mole of enzyme subunit. The denatured enzyme contains 10 sulfhydryl groups per subunit, as shown after reaction with 5,5'-dithio-bis-(2-nitrobenzoic acid), of which 5 can be titrated also in the native enzyme, exhibiting time-dependent reactivity. One sulfhydryl group is located in the coenzyme binding site. This study shows that the physical and catalytic properties of P. stutzeri MDH strongly resemble those of the mitochondrial eukariotic enzyme. This finding strengthens the existing view that, in the evolution process, the mitochondrial MDH might have appeared before the cytoplasmic.
authors.affiliation: Department of Agricultural Biotechnology, The Agricultural University of Athens, Greece.
authors.forename: N E
authors.initials: NE
authors.lastname: Labrou
authors.forename: Y D
authors.initials: YD
authors.lastname: Clonis
mesh_terms: D000345:Affinity Labels; D000880:Anthraquinones / Q000378:metabolism; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002852:Chromatography, Ion Exchange; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D004734:Energy Metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D011549:Pseudomonas / Q000201:enzymology*; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013816:Thermodynamics; D014227:Triazines / Q000378:metabolism
chemical_list: D000345:Affinity Labels; D000880:Anthraquinones; D013438:Sulfhydryl Compounds; D014227:Triazines; C091762:Vilmafix Blue A-R; D004228:Dithionitrobenzoic Acid; D008291:Malate Dehydrogenase