Contributions of binding and catalytic rate constants to evolutionary modifications inK m of NADH for muscle-type (M4) lactate dehydrogenases

Abstract

The apparent Michaelis constant (Km) of NADH for muscle-type (M4 isozyme) lactate dehydrogenases (LDHs) is highest, at any given temperature of measurement, for LDHs of cold-adapted vertebrates (Table 1). However, these interspecific differences in theKm of NADH are not due to variations in LDH-NADH binding affinity. Rather, theKm differences result entirely from interspecific variation in the substrate turnover constant (kcat) (Fig. 1; Table 2). This follows from the fact that theKm of NADH is equal tokcat divided by the “on constant” for NADH binding to LDH,k1, so that interspecific differences inkcat, combined with identical values fork1 among different LDH reactions, make the magnitude of theKm of NADH a function of substrate turnover number. The temperature dependence of theKm of NADH for a single LDH homologue is the net result of temperature dependence of bothkcat andk1 (Figs. 3 and 4). Temperature independentKm values can result from simultaneous, and algebraically offsetting, increases inkcat andk1 with rising temperature. Salt-induced changes in theKm of NADH also may be due to simultaneous perturbation of bothkcat andk1 (Table 3). These findings are discussed from the standpoint of the evolution of LDH kinetic properties, particularly the interspecific conservation of catalytic and regulatory functions, in differently-adapted species.