Enzymological characteristics of the hyperthermostable NAD-dependent glutamate dehydrogenase from the archaeon Pyrobaculum islandicum and effects of denaturants and organic solvents.
NAD-dependent glutamate dehydrogenase (L-glutamate:NAD oxidoreductase, deaminating; EC 1.4.1.2) was purified to homogeneity from a crude extract of the continental hyperthermophilic archaeon Pyrobaculum islandicum by two successive Red Sepharose CL-4B affinity chromatographies. The enzyme is the most thermostable NAD-dependent dehydrogenase found to date; the activity was not lost after incubation at 100 degrees C for 2 h. The enzyme activity increased linearly with temperature, and the maximum was observed at ca. 90 degrees C. The enzyme has a molecular mass of about 220 kDa and consists of six subunits with identical molecular masses of 36 kDa. The enzyme required NAD as a coenzyme for L-glutamate deamination and was different from the NADP-dependent glutamate dehydrogenase from other hyperthermophiles. The Km values for NAD, L-glutamate, NADH, 2-oxoglutarate, and ammonia were 0.025, 0.17, 0.0050, 0.066, and 9.7 mM, respectively. The enzyme activity was significantly increased by the addition of denaturants such as guanidine hydrochloride and some water-miscible organic solvents such as acetonitrile and tetrahydrofuran. When fluorescence of the enzyme was measured in the presence of guanidine hydrochloride, a significant emission spectrum change and a shift in the maximum were observed but not in the presence of urea. These results indicate that this hyperthermophilic enzyme may have great potential in applications to biosensor and bioreactor processes.
Kujo
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C
Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, Tokushima 770-8506, Japan.
Ohshima
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T
D000595:Amino Acid Sequence; D003902:Detergents / Q000494:pharmacology; D004795:Enzyme Stability; D005969:Glutamate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019791:Guanidine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D011489:Protein Denaturation; D017386:Sequence Homology, Amino Acid; D012997:Solvents / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature; D019624:Thermoproteaceae / Q000201:enzymology* / Q000235:genetics
D003902:Detergents; D012997:Solvents; D005969:Glutamate Dehydrogenase; D019791:Guanidine