CHARACTERIZATION OF AN EXTREMELY THERMOSTABLE GLUTAMATE-DEHYDROGENASE - A KEY ENZYME IN THE PRIMARY METABOLISM OF THE HYPERTHERMOPHILIC ARCHAEBACTERIUM, PYROCOCCUS-FURIOSUS

Glutamate dehydrogenase (L-glutamate:NAD(P)+ oxidoreductase, deaminating, EC 1.4.1.3) from the hyperthermophilic Archeon Pyrococcus furiosus was purified to homogeneity by chromatography on anion-exchange, molecular-exclusion and hydrophobic-interaction media. The purified native enzyme had an M(r) of 270 000 +/- 15 000 and was shown to be a hexamer with identical subunits of M(r) 46 000. The enzyme was exceptionally thermostable, having a half-life of 3.5 to more than 10 h at 100-degrees-C, depending on the concentration of enzyme. The K(m) of the enzyme for ammonia was high (9.5 mM), indicating that the enzyme is probably active in the deaminating, catabolic direction. The coenzyme utilization of the enzyme resembled the equivalent enzymes from eukaryotes rather than eubacteria, since both NADH and NADPH were recognized with high affinity. The enzyme displayed a preference for NADP+ over NAD+ that was more pronounced at low assay temperatures (50-70-degrees-C) compared with the optimal temperature for enzyme activity, 95-degrees-C.