title: Subunit interactions in mitochondrial malate dehydrogenase. Kinetics and mechanism of reassociation.
abstract: The pH-dependent dissociation of porcine heart mitochondrial malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) has been more extensively characterized. The native, dimeric form of the enzyme (Mr = 70,000) which exists at pH 7.5 has previously been shown to dissociate into its constituent subunits (Mr = 35,000) at pH 5.0 (Bleile, D. M., Schulz, R. A., Gregory, E. M., and Harrison, J. H. (1977) J. Biol. Chem. 252, 755-758). The dissociation is accompanied by a concomitant decrease in enzymatic specific activity and an increase in intrinsic protein fluorescence. By using the characteristics of specific activity and intrinsic protein fluorescence as probes of dimerization, the kinetics of subunit reassociation was investigated. In order to facilitate reassociation, a pH jump method was utilized in which enzyme at pH 5.0 was diluted into a large excess of pH 7.5 buffer. The regain of enzymatic specific activity and the decrease in protein fluorescence were observed to follow first order kinetics. The rate constant in both cases was dependent upon the protein concentration, and in all cases, full recovery of either enzymatic activity or native protein fluorescence was obtained. The Arrhenius activation energy for the reassociation of the subunits was found to be approximately 20 kcal/mol, an observation which is consistent with a refolding process whose rate-limiting step may be the cis/trans-isomerization about one or more proline imino bonds. A model for subunit reassociation which is consistent with the kinetic data is proposed.
mesh_terms: D000818:Animals; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008291:Malate Dehydrogenase / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000201:enzymology*; D009243:NAD; D010084:Oxidation-Reduction; D013050:Spectrometry, Fluorescence; D013552:Swine; D013696:Temperature
chemical_list: D046911:Macromolecular Substances; D009243:NAD; D008291:Malate Dehydrogenase