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 Km 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.
Robb
F T
FT
Center of Marine Biotechnology, University of Maryland, Baltimore 21202.
Park
J B
JB
Adams
M W
MW
D000595:Amino Acid Sequence; D001105:Archaea / Q000737:chemistry / Q000201:enzymology*; D002384:Catalysis; D004586:Electrophoresis; D004795:Enzyme Stability; D005969:Glutamate Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013379:Substrate Specificity
D005969:Glutamate Dehydrogenase