Amino acid residues involved in the catalytic mechanism of NAD-dependent glutamate dehydrogenase from Halobacterium salinarum

The pH dependence of kinetic parameters for a competitive inhibitor (glutar ate) was determined in order to obtain information on the chemical mechanis m for NAD-dependent glutamate dehydrogenase from Halobacterum salinarum. Th e maximum velocity is pH dependent, decreasing at low pHs giving a pK value of 7.19 +/- 0.13, while the V/K for L-glutamate at 30 degrees C decreases at low and high pHs, yielding pK values of 7.9 +/- 0.2 and 9.8 +/- 0.2, res pectively. The glutarate pK(is), profile decreases at high pHs, yielding a pK of 9.59 +/- 0.09 at 30 degrees C. The values of ionization heat calculat ed from the change in pK with temperature are: 1.19 x 10(4), 5.7 x 10(3), 7 x 10(3), 6.6 x 10(3) cal mol(-1), for the residues involved. All these dat a suggest that the groups required for catalysis and/or binding are lysine, histidine and tyrosine. The enzyme shows a time-dependent loss in glutamat e oxidation activity when incubated with diethyl pyrocarbonate (EPC). Inact ivation follows pseudo-first-order kinetics with a second-order rate consta nt of 53 M-1 min(-1). The pK(a) of the titratable group was pK(1) = 6.6 +/- 0.6. Inactivation with ethyl acetimidate also shows pseudo-first-order kin etics as well as inactivation with TNM yielding second-order constants of 1 .2 M-1 min(-1) and 2.8 M-1 min(-1), and pK(a)s of 8.36 and 9.0, respectivel y. The proposed mechanism involves hydrogen binding of each of the two carb oxylic groups to tyrosyl residues; histidine interacts with one of the N-hy drogens of the L-glutamate amino group. We also corroborate the presence of a conservative lysine that has a remarkable ability to coordinate a water molecule that would act as general base. (C) 1999 Elsevier Science B.V. All rights reserved.