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 ext ract of the continental hyperthermophilic archaeon Pyrobaculum islandi cum by two successive Red Sepharose CL-4B affinity chromatographies, T he enzyme is the most thermostable NAD-dependent dehydrogenase found t o date; the activity was not lost after incubation at 100 degrees C fo r 2 h, The enzyme activity increased linearly with temperature, and th e 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 mol ecular masses of 36 kda. The enzyme required NAD as a coenzyme for L-g lutamate deamination and was different from the NADP-dependent glutama te dehydrogenase from other hyperthermophiles. The K-m values for NAD, L-glutamate, NADH, 2-oxoglutarate, and ammonia were 0.025, 0.17, 0.00 50, 0.066, and 9.7 mM, respectively, The enzyme activity was significa ntly increased by the addition of denaturants such as guanidine hydroc hloride and some water-miscible organic solvents such as acetonitrile and tetrahydrofuran, When fluorescence of the enzyme was measured in t he presence of guanidine hydrochloride, a significant emission spectru m change and a shift in the maximum were observed but not in the prese nce of urea. These results indicate that this hyperthermophilic enzyme may have great potential in applications to biosensor and bioreactor processes.