PURIFICATION AND CHARACTERIZATION OF A NOVEL ADP-DEPENDENT GLUCOKINASE FROM THE HYPERTHERMOPHILIC ARCHAEON PYROCOCCUS-FURIOSUS

Pyrococcus furiosus uses a modified Embden-Meyer-hof pathway during gr owth on poly- or disaccharides. Instead of the usual ATP-dependent glu cokinase, this pathway involves a novel ADP-dependent (AMP-forming) gl ucokinase. The level of this enzyme and some other glycolytic enzymes appeared to be closely regulated by the substrate, Growth on cellobios e resulted in a high specific activity of 0.96 units mg(-1), whereas o n pyruvate a 10-fold lower activity was found, The ADP-dependent gluco kinase was purified 1350 fold to homogeneity. The oxygen-stable enzyme had a molecular mass of 93 kDa and was composed of two identical subu nits (47 kDa). The glucokinase was highly specific for ADP, which coul d not be replaced by ATP, phosphoenolpyruvate, GDP, PPi, or polyphosph ate, D-Glucose could be replaced only by a-deoxy-D-glucose, albeit wit h a low efficiency, The K-m values for D-glucose and ADP were 0.73 and 0.033 mM, respectively, An optimum temperature of 105 degrees C and a half-life of 220 min at 100 degrees C are in agreement with the requi rements of this hyperthermophilic organism. The properties of the gluc okinase are compared to those of less thermoactive gluco/hexokinases.