Binding of reduced and oxidized nicotinamide adenine dinucleotide to pig heart supernatant malate dehydrogenase

Abstract

The association reactions of NADH and NAD+ with dimeric pig heart supernatant malate dehydrogenase (s-MDH) have been measured at pH 6 and 8 by calorimetric and fluorescence methods, and the thermodynamic parameters describing these reactions have been evaluated. Coenzyme binding is associated with the uptake of 0.55 mol of H+/mol of NADH at pH 8 and 0.19 mol of H+ at pH 6. No significant effect of NAD+ binding on proton binding was observed. Increase in ionic strength strongly affects the free energies of binding of NAD+ and NADH. No cooperativity was observed in the enthalpy or free energy changes for binding of NAD+ or NADH. The differences in free energy of binding of NAD+ and NADH and the effect of pH on binding of NADH are entropy based. These effects are interpreted as reflecting a small number of interactions within the active site that are predominantly ionic.