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Thermodynamic studies of binary and ternary complexes of pig-skeletal-muscle lactate dehydrogenase.

The thermodynamics of formation of the various intermediary complexes in the reaction catalyzed by pig skeletal muscle lactate dehydrogenase (EC 1.1.1.27) has been investigated in 0.2 M potassium phosphate buffer, pH 7, over the temperature range 10–36°C. As with the analogous experiments on pig heart muscle lactate dehydrogenase [Schmid, F., Hinz, H.-J. & Jaenicke, R. (1976) Biochemistry, 15, 3052–3059] the catalytic process was approximated by employing the substrate-analogous inhibitors oxamate and oxalate instead of pyruvate and lactate, respectively, in the two reactions forming the ternary complexes lactate dehydrogenase · NADH · oxamate and lactate dehydrogenase · NAD · oxalate. The standard Gibbs free energy for association of oxamate with the binary lactate dehydrogenase · NADH complex has been derived from fluorescence titrations, while the corresponding value for the binding of oxalate to the lactate dehydrogenase · NAD complex resulted from ultraviolet difference spectroscopy studies. All reaction enthalpies stem from direct calorimetric measurements. The prominent feature of the calorimetric studies is the pronounced variation with temperature of binding enthalpies, showing a negative temperature coefficient of −1901 ± 46 (− 7954 ± 193) cal (J) · (mol of enzyme)−1· K−1 for the association of oxamate and the lactate dehydrogenase · NADH complex and of −1484 ± 80 (6209 ± 335) cal (J) · (mol of enzyme)−1· K−1 for the reaction between lactate dehydrogenase · NAD and oxalate. These negative Δcp values involved in the binding reactions are suggestive of structural alterations of the enzyme induced by ligand binding, which consist of a limited immobilization of the protein with respect to vibrational modes of freedom and/or partial reversal of hydrophobic hydration. These effects are somewhat larger for the pig skeletal muscle enzyme than for the pig heart muscle enzyme.