The anomalous properties of the glutamate dehydrogenase-NADPH-alpha-ketoglutarate complex are not ascribable to a carbonyl addition reaction

• Published in: The Journal of biological chemistry 1987-08, Vol.262 (24), p.11684-11687
• Main Authors: Fisher, H F, Pazhanisamy, S, Medary, R T
• Format: Article
• Language: English
• Subjects: alpha -ketoglutaric acid; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Cattle; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; glutamate dehydrogenase; Glutamate Dehydrogenase (NADP+); Glutamate Dehydrogenase - metabolism; Ketoglutaric Acids - metabolism; Kinetics; Liver - enzymology; NADPH; Oxidoreductases; Spectrophotometry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)60864-6

The glutamate dehydrogenase-NADPH-alpha-ketoglutarate complex, an active intermediate on the reaction pathway has a number of unusual properties: 1) it is the only blue-shifted natural complex of this enzyme; 2) it has an anomalously slow rate of dissociation; 3) its off-rate shows a substantial pH-independent D2O solvent isotope effect not exhibited by any other ternary complex of this enzyme; and 4) it has an unusually large enthalpy of interaction parameter. These properties must be ascribable to at least one of the two possibilities conferred on the complex by the presence of the alpha-carbonyl group of alpha-ketoglutarate; the ability to engage in carbonyl addition reactions; and/or the ability to form a specific hydrogen bond. Oxalylglycine, a competitive inhibitor of alpha-ketoglutarate in this enzyme-catalyzed reaction, provides a means of discriminating between these two modes of action. The structure of oxalylglycine provides a dicarboxylic compound which has the same intercarboxylate proton distance and has a carbonyl group in a position spatially analogous to that of alpha-ketoglutarate. Its carbonyl group, however, is that of an amide group and cannot, therefore, engage in carbonyl addition reactions, but can hydrogen bond. Therefore, any effects observed with both oxalylglycine and alpha-ketoglutarate must be ascribed to formation of specific alpha-carbonyl hydrogen bonding, whereas any effects observed with alpha-ketoglutarate alone must be due to an alpha-carbonyl addition reaction. We have used this logic to test the source of the four phenomena listed above. In each case, oxalylglycine and alpha-ketoglutarate showed the same effect. Therefore, we conclude that all four phenomena are in fact due to the formation of a specific alpha-carbonyl hydrogen bond and that the specific carbonyl addition reaction between alpha-ketoglutarate and an enzyme lysine group, postulated in one proposed catalytic mechanism, does not occur.