Ml. Camacho et al., NAD-GLUTAMATE DEHYDROGENASE FROM HALOBACTERIUM-HALOBIUM - PH AND CHEMICAL MODIFICATION STUDIES, International Journal of Biochemistry, 25(7), 1993, pp. 979-985
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.