Coenzyme binding ability of homologs of M4-lactate dehydrogenase in temperature adaptation

Temperature effects on dissociation constants (Kd), binding enthalpies and apparent Michaelis constants (Km) for NADH, plus Arrhenius activation energies (Ea), substrate turnover numbers (kcat), and NADH 'on' constants (k1) were measured or calculated for M4-lactate dehydrogenase homologs from deep-sea, midwater, shallow-water temperate, and shallow-water tropical teleost fishes, and a mammal. At any single measurement temperature, Km and kcat values were significantly higher for groups adapted to lower temperatures. This pattern of Km values and temperature illustrates a strong evolutionary conservation of Km of NADH. When determined at the average body temperature of each species, the Km values are very similar, resulting in the preservation of the catalytic capacity and regulatory properties of these enzyme homologs at their in situ temperatures. In contrast, Kd values, while varying considerably among species, are not significantly different among the different groups at any one temperature. The ratio of Km to Kd tends to follow a phylogenetic pattern rather than a pattern of environmental adaptation. Thus, evolutionary adjustments in Km are not directly the result of changes in cofactor binding. All the rate constants involved in determining the Km and Kd of NADH (kcat, k1 and k-1) can be modified.