The effect of ligands on the irreversible inhibition of the NAD+-dependent isocitrate dehydrogenase from ox brain.

The effects of ligands on the irreversible inhibition of the NAD+-dependent isocitrate dehydrogenase from ox brain were studied. Isocitrate in the presence of Mg2+ ions was found to protect against denaturation at 40 degrees C and this protection was enhanced by ADP which also protected on its own. None of the substrates or the activator ADP afforded protection against inhibition by diethylpyrocarbonate. Inactivation by this compound and by elevated temperatures did not obey first-order kinetics with respect to time. Inhibition by iodoacetate appeared to obey first-order kinetics both with respect to time and to inhibitor concentration. Protection against inhibition was afforded by isocitrate, NAD+ and ADP. The dependence of the extent of protection on the concentration of the magnesium-isocitrate complex was sigmoid at both pH 6.5 and pH 7.5 indicating the apparent homotropic cooperativity seen in initial-rate kinetic experiments to be a reflection of cooperative binding of this substrate. ADP reduced the affinity of the enzyme for this substrate without affecting the degree of this cooperativity. The dependence of the extent of protection by ADP upon its concentration obeyed Michaelis-Menten kinetics. The significance of these observations in terms of the kinetic and allosteric mechanism followed by this enzyme is discussed in the light of previous kinetic studies.