Thermodynamic studies of binary and ternary complexes of pig heart lactate dehydrogenase.
The thermodynamics of the reaction catalyzed by pig heart muscle lactate dehydrogenase (LDH; EC1.1.1,27) have been studied in 0,2 M potassium phosphate buffer, pH 7, over the temperature range of 10 to 35 degrees C by using oxamate and oxalate to simulate the corresponding reactions of the substrates pyruvate and lactate, respectively. The various complexes formed are characterized by Gibbs free energies, enthalpies, and entropies. The Gibbs free energies were determined by equilibrium dialysis investigations, fluorescence titrations, and ultraviolet difference spectroscopy, while the reaction enthalpies stem from direct calorimetric measurements, Formulas are given for both the temperature dependence of the equilibrium constants and the variation with temperature of the enthalpies involved in the four reactions between LDH and NADH or NAD, LDH-NADH and oxamate, and LDH-NAD and oxalate. All reactions show a marked negative temperature coefficient, deltacp, of the binding enthalpies indicating partial refolding to be associated with binary and ternary complex formation. This interpretation appears very probable in view of recent x-ray crystallographic studies on lactate dehydrogenase from dogfish, which demonstrate a volume decrease to occur on binding of oxamate to the LDH-NADH complex. The validity of the thermodynamic parameters, as derived with substrate analogues, for the actual catalytic reaction, gains strong support from the agreement between the sum of the heats involved in the four intermediary reactions reported in this study and direct determinations of the overall enthalpy associated with the catalytic process published in the literature.
D000818:Animals; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D009206:Myocardium / Q000201:enzymology; D009243:NAD / Q000378:metabolism; D010070:Oxalates / Q000494:pharmacology; D010072:Oxamic Acid / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D013552:Swine; D013696:Temperature; D013816:Thermodynamics
D010070:Oxalates; D009243:NAD; D007770:L-Lactate Dehydrogenase; D010072:Oxamic Acid