Regulation of Mitochondrial Glutamic Dehydrogenase by Divalent Metals, Nucleotides, and α-Ketoglutarate

The substrates, NAD+, NADH, and to a lesser extent, glutamate, of a freely reversible glutamic dehydrogenase from Blastocladiella emersonii, and diverse allosteric modulators (AMP, ADP, Ca++, Mn++) have been shown to bind to the enzyme and prevent it from dissociating into monomers at alkaline pH, high ionic strength, and under hydrodynamic shear. At a fixed pH and ionic strength, these ligands display varying magnitude of effectiveness as follows: (AMP, Ca++, NAD+, NADH) g Mn++ g glutamate. The enzyme dissociates irreversibly from an active tetramer (mol wt 200,000 ± 21,000) to inactive monomers (48,500 ± 7,000 daltons). Metabolites and ethylenediaminetetraacetate which have been shown to inhibit the enzyme unidirectionally (LeJohn, H. B., J. Biol. Chem., 243, 5126 (1968)) enhance the dissociation effects of pH and hydrodynamic shear. The inhibitory effects of Cu++ and Hg++ proved useful since they are the only metal ligands found that could significantly inhibit the oxidation-reduction reactions of the enzyme, and Cu++ dissociates the enzyme into monomers at all pH values between 6 and 9, but its disruptive effects could be reversed at alkaline pH values by the allosteric modulators. Product inhibition studies have shown that in the presence of AMP, the mechanism of enzyme action is ordered binary-ternary (by Cleland's designation, Biochim. Biophys. Acta, 67, 104, 173 (1963)). Extensive zone sedimentation studies of the molecular state of the enzyme under a variety of conditions, and close correlations with kinetic patterns suggest the following. (a) The cooperative patterns of substrate-enzyme interaction is explicable in terms of association and dissociation of the enzyme. (b) Ca++ may have important physiological functions in regulating the flow of metabolites across the mitochondrial membrane. (c) α-Ketoglutarate may act as an allosteric inhibitor of glutamate oxidation. No other reactant or product displays this cooperative property. This allosteric effect of α-ketoglutarate on glutamate oxidation has been utilized, in conjunction with kinetic and physiological data, to explain how carbon can flow reversibly into organic and amino acids through the action of Blastocladiella glutamic dehydrogenase.