Cooperativity in the mechanism of malate dehydrogenase.

Abstract

Cooperativity in the catalytic mechanism of porcine cytoplasmic malate dehydrogenase (sMDH) has been a point of ongoing discussion. Though previous investigations revealed little evidence of cooperativity, chemical modification studies reported by this laboratory demonstrate that binding of cofactor or cofactor plus substrate causes the enzyme's subunits to become chemically nonidentical. Therefore, we have reexamined the enzyme's steady-state kinetic and ligand-binding properties. To aide in characterizing sMDH kinetics, activities of the native enzyme and of sMDH, which was partially inactivated by an active-site-specific reagent, were examined. As expected for a negatively cooperative enzyme, steady-state kinetics (at pH 8.0, the pH optimum of the enzyme) are characterized by concave Eadie-Hofstee plots. Further, qualitative as well as quantitative results from partially inactivated sMDH strongly support negative cooperativity and eliminate many alternative mechanisms. Finally, results from equilibrium binding experiments are consistent with cytoplasmic malate dehydrogenase binding NADH in a negatively cooperative manner. Together, these results indicate that cytoplasmic malate dehydrogenase acts as a negatively cooperative enzyme.