The effect of pH on the allosteric behaviour of ox-brain NAD+-dependent isocitrate dehydrogenase

The sigmoid dependence of the initial rate of the reaction catalysed by NAD+-dependent isocitrate dehydrogenase on the concentration of isocitrate is greatly reduced as the pH is decreased. The apparent pK for this process is 7.3. At pH values of 6.0 and 6.5 the enzyme exhibits hyperbolic kinetics with respect to isocitrate at relatively high concentrations of NAD+, but the dependence becomes sigmoid at lower NAD+ concentrations. The allosteric activators ADP and citrate have only small effects on the activity of the enzyme at pH 6.5 but in the latter case the effects are increased by decreasing the concentration of NAD+. The dependence of initial velocity on the NAD+ concentration is hyperbolic at all pH values in the pH range 6--8.5. NADH is a competitive inhibitor of enzyme activity with respect to NAD+, and its presence induces a sigmoid dependence of initial velocity on isocitrate concentration at pH 6.5 and in the presence of high concentrations of NAD+. Kinetic studies at pH 6.5 indicate that the apparent maximum velocity of the reaction with respect to NAD+ is insensitive to changes in the isocitrate concentration and that with isocitrate as the substrate is relatively insensitive to changes in the NAD+ concentration under conditions where the behaviour appears to be hyperbolic. threo-Ds-Isocitrate is the true substrate for the enzyme and the corresponding Ls isomer is neither a substrate nor an inhibitor.