Enzyme Variants in Thermal Acclimation

Abstract

Abstract Citrate synthase occurs in two kinetically distinguishable forms, termed C and W in cold and warm acclimated trout, respectively. Oxalacetate and acetyl-CoA saturation curves for both enzymes are hyperbolic and do not show any substrate inhibition. The apparent Km values for the C form increase with temperature above 10°. At saturating (0.1 mm) levels of oxalacetate, the Km of acetyl-CoA is 0.05 mm at 10°, about one-fifth the value at 35°. At saturating levels of acetyl-CoA, the Km of oxalacetate is 7 µm at 10°, about one-fourth the value at 35°. These values at low temperature are similar to the Km constants for the W form below 15°, but above 15°, the Km values for the W form are distinctly lower than for the C form. In the upper biological temperature range for both cold and warm acclimated trout, the Q10 for citrate synthase is about 1.0 at low substrate concentrations because the increase in Km compensates for the increasing thermal energy. At saturating substrate concentrations, the temperature characteristics of the two enzymes are the same, and the calculated activation energies are 8.8 kcal per mole. Both C and W forms of citrate synthase are strongly inhibited by ATP, which increases the Km of acetyl-CoA (5-fold at pH 7.5 and 22°) but does not alter the calculated Vmax. ATP inhibition is noncompetitive with respect to oxalacetate, and the oxalacetate saturation curve remains hyperbolic in the presence of ATP. The Ki of ATP for the C form increases dramatically with temperature above 15°, while the Ki of the W form is thermally insensitive to nearly the lethal limit for trout. The calculated Vmax values for both C and W forms increase 4-fold between pH 6.5 and 8.5, but the Km values are rather insensitive to pH changes. The Ki of ATP, on the other hand, increases 4-fold between pH 6.5 and 8.5. Since the pH in poikilotherms increases at low temperature, these effects of pH on citrate synthase may (a) reduce the Q10 of the reaction irrespective of substrate concentrations, and (b) reduce thermal effects on ATP inhibition.