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 deamin ation Of L-glutamate (glutamate oxidation) and for the amination of 2- oxoglutarate (glutamate synthesis) catalyzed by halophilic NAD-glutama te dehydrogenase has been determined with the aim of elucidating the r ole that ionizing amino acid residues play in binding and catalysis. 2 . In the deamination reaction two possible groups on the enzyme with p Ks of 9.07 and 9.67 at 40-degrees-C must be unprotonated and protonate d, 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 activit y. 4. In the amination reaction, the analysis of these data revealed t hat two enzyme groups are required for catalysis and/or substrate bind ing 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 w ith temperature, values of 11,947 and 7,392 cal/mol have been calculat ed for the heats of ionization of the deamination and amination reacti ons, respectively. 6. All these data suggest that the imidazole group of a histidine residue and/or the epsilon-amine group of a lysine resi due could participate in catalysis and/or binding. 7. Chemical modific ation of halophilic NAD-glutamate dehydrogenase with diethyl pyrocarbo nate, a histidine-modifying agent, has suggested that a single histidi ne residue is involved in the binding and/or catalysis of the enzyme.