title: Determination of the catalytic mechanism for mitochondrial malate dehydrogenase.
abstract: The kinetics of malate dehydrogenase (MDH) catalyzed oxidation/reduction of L-malate/oxaloacetate is pH-dependent due to the proton generated/taken up during the reaction. Previous kinetic studies on the mitochondrial MDH did not yield a consensus kinetic model that explains both substrate and pH dependency of the initial velocity. In this study, we propose, to our knowledge, a new kinetic mechanism to explain kinetic data acquired over a range of pH and substrate concentrations. Progress curves in the forward and reverse reaction directions were obtained under a variety of reactant concentrations to identify associated kinetic parameters. Experiments were conducted at physiologically relevant ionic strength of 0.17 M, pH ranging between 6.5 and 9.0, and at 25 °C. The developed model was built on the prior observation of proton uptake upon binding of NADH to MDH, and that the MDH-catalyzed oxidation of NADH may follow an ordered bi-bi mechanism with NADH/NAD binding to the enzyme first, followed by the binding of oxaloacetate/L-malate. This basic mechanism was expanded to account for additional ionic states to explain the pH dependency of the kinetic behavior, resulting in what we believe to be the first kinetic model explaining both substrate and pH dependency of the reaction velocity.
authors.affiliation: Department of Molecular and Integrated Physiology, University of Michigan, Ann Arbor, Michigan.
authors.forename: Santosh K
authors.initials: SK
authors.lastname: Dasika
authors.affiliation: Department of Molecular and Integrated Physiology, University of Michigan, Ann Arbor, Michigan.
authors.forename: Kalyan C
authors.initials: KC
authors.lastname: Vinnakota
authors.affiliation: Department of Molecular and Integrated Physiology, University of Michigan, Ann Arbor, Michigan. Electronic address: beardda@umich.edu.
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Beard
mesh_terms: D000818:Animals; D055162:Biocatalysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000737:chemistry* / Q000378:metabolism; D008956:Models, Chemical; D009243:NAD / Q000378:metabolism; D011485:Protein Binding; D013552:Swine
chemical_list: D024101:Mitochondrial Proteins; D009243:NAD; D008291:Malate Dehydrogenase