Association-Dissociation Reactions of Mitochondrial Isocitric Dehydrogenase Induced by Protons and Various Ligands

Abstract

A nicotinamide adenine dinucleotide-specific isocitric dehydrogenase has been isolated and purified 450-fold from Blastocladiella emersonii. The enzyme has a monomeric molecular weight of 110,000 ± 10,000. It is capable of aggregating to polymers of molecular weight ranging from 200,000 to 500,000 and higher at acid pH levels. At alkaline pH values, it exists as a monomer. The aggregated enzyme is inactive and the monomer is active. Under the influence of various ligands (citrate, isocitrate, Mn++, Mg++), the aggregated enzyme can be converted to the monomer. A difference of 1 pH unit (6.5 to 7.5) changes the association-dissociation equilibrium in favor of monomer formation from the polymeric form. The enzyme was shown to exist as two distinct ionized forms with pH optima at 6.5 and 9.0. By a complex series of ligand interactions over a wide range of pH values, the two pH optima disappear and a single ionized species becomes evident with a pH optimum at 8. Although isocitrate and NAD+ display cooperative interactions, at low pH levels and high NAD+ concentrations, NAD+ is incapable of dissociating the polymer into monomer whereas isocitrate can. The same incompetence is shown by AMP but not by citrate, both of which are allosteric activators. Kinetic analyses have shown that the kinetic mechanism may be ordered binary-ternary. Attempts have been made to relate these and other findings to a system of regulation by which this enzyme is coupled to the glutamic dehydrogenase found in the mitochondria. Very tenuous correlations have been proposed for the participation of this regulatory mechanism in the germination process in this organism.