NAD-GLUTAMATE DEHYDROGENASE FROM HALOBACTERIUM-HALOBIUM - PH AND CHEMICAL MODIFICATION STUDIES

1. The pH variation of the kinetic parameters V and V/K for the deamination Of L-glutamate (glutamate oxidation) and for the amination of 2-oxoglutarate (glutamate synthesis) catalyzed by halophilic NAD-glutamate dehydrogenase has been determined with the aim of elucidating the role that ionizing amino acid residues play in binding and catalysis. 2. In the deamination reaction two possible groups on the enzyme with pKs of 9.07 and 9.67 at 40-degrees-C must be unprotonated and protonated, respectively, for NAD+ binding. 3. The V/K profile for L-glutamate shows that a group with a pK around 8 must be unprotonated for activity. 4. In the amination reaction, the analysis of these data revealed that two enzyme groups are required for catalysis and/or substrate binding with pK values of approx 6.5 and 10.0 at 40-degrees-C that must be unprotonated and protonated, respectively. 5. From the change in pK with temperature, values of 11,947 and 7,392 cal/mol have been calculated for the heats of ionization of the deamination and amination reactions, respectively. 6. All these data suggest that the imidazole group of a histidine residue and/or the epsilon-amine group of a lysine residue could participate in catalysis and/or binding. 7. Chemical modification of halophilic NAD-glutamate dehydrogenase with diethyl pyrocarbonate, a histidine-modifying agent, has suggested that a single histidine residue is involved in the binding and/or catalysis of the enzyme.