Interrelations between pH and temperature for the catalytic rate of the M4 lsozyme of lactate dehydrogenase (EC 1.1.1.27) from goldfish (Carassius auratus L.).

A kinetic study of the rate of pyruvate reduction by goldfish LDH-M₄ (the homotetrameric form of lactate dehydrogenase which predominates in skeletal muscle) provided an analysis of the effects of pH and temperature on v (reaction velocity) and estimates of how temperature might affect catalysis in vivo, where the physiological pH regulation imposes a temperature coefficient of ?0.015 to ?0.020 pH unit/ °C. Consistent with published data for other LDHs, (i) V (maximum reaction velocity) was pH insensitive over a physiological pH range, (ii) the K_m for pyruvate, K_P, was sensitive to both pH and temperature, and (iii) the K_m for NADH and the dissociation constant for NADH were both sensitive to temperature, but not to pH. V approximately doubled with each 10 °C (E_a = 11.7 kcal/mol). The effects of pH and temperature on K_P were consistent with two enthalpic contributions, an ionization enthalpy (ΔH_i^°) of 7.2 kcal/mol (probably a histidine imidazole), and an enthalpy (ΔH_SO) of 5.8 kcal/mol for the combination of pyruvate with the nonionized (pH ? pK′) LDH-NADH complex. When the pH was varied according to the physiological temperature coefficient, v was more sensitive to temperature than for conditions of constant pH, the usual design of kinetic experiments. This effect was due to the decreased temperature sensitivity of K_P caused by partial concellation of the ΔH_i^° effect by the pH regulation: $\text{d}\text{pH}\text{dT}\text{?}\text{d}\text{p}\text{K′}\text{dT}$. At constant pH, on the other hand, K_P increased strongly with temperature and had the effect of offsetting (especially at higher pH values) the large increases in V. It was suggested that the magnitudes of ΔH_i^° and ΔH_SO might have been important in the evolution of LDHs and other enzymes of cold-blooded animals.