Purification and characterization of a novel ADP-dependent glucokinase from the hyperthermophilic archaeon Pyrococcus furiosus

Abstract

Pyrococcus furiosus uses a modified Embden-Meyerhof pathway during growth on poly- or disaccharides. Instead of the usual ATP-dependent glucokinase, this pathway involves a novel ADP-dependent (AMP-forming) glucokinase. The level of this enzyme and some other glycolytic enzymes appeared to be closely regulated by the substrate. Growth on cellobiose resulted in a high specific activity of 0.96 units mg-1, whereas on pyruvate a 10-fold lower activity was found. The ADP-dependent glucokinase was purified 1350-fold to homogeneity. The oxygen-stable enzyme had a molecular mass of 93 kDa and was composed of two identical subunits (47 kDa). The glucokinase was highly specific for ADP, which could not be replaced by ATP, phosphoenol-pyruvate, GDP, PP(i), or polyphosphate. D-Glucose could be replaced only by 2-deoxy-D- glucose, albeit with 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 °C and a half-life of 220 min at 100 °C are in agreement with the requirements of this hyperthermophilic organism. The properties of the glucokinase are compared to those of less thermoactive gluco/hexokinases.