Molecular and biochemical characterization of the ADP-dependent phosphofructokinase from the hyperthermophilic archaeon Pyrococcus furiosus

Pyrococcus furiosus uses a modified Embden-Meyerhof pathway involving two A DP-dependent kinases. Using the N-terminal amino acid sequence of the previ ously purified ADP-dependent glucokinase, the corresponding gene as well as a related open reading frame were detected in the genome of P. furiosus. B oth genes were successfully cloned and expressed in Escherichia coli, yield ing highly thermoactive ADP-dependent glucokinase and phosphofructokinase. The deduced amino acid sequences of both kinases were 21.1% identical but d id not reveal significant homology with those of other known sugar kinases. The ADP-dependent phosphofructokinase was purified and characterized. The oxygen-stable protein had a native molecular mass of approximately 180 kDa and was composed of four identical 52-kDa subunits. It had a specific activ ity of 88 units/mg at 50 degrees C and a pH optimum of 6.5. As phosphoryl g roup donor, ADP could be replaced by GDP, ATP, and GTP to a limited extent. The K-m values for fructose 6-phosphate and ADP were 2.3 and 0.11 mM, resp ectively. The phosphofructokinase did not catalyze the reverse reaction, no r was it regulated by any of the known allosteric modulators of ATP depende nt phosphofructokinases. ATP and AMP were identified as competitive inhibit ors of the phosphofructokinase, raising the K-m for ADP to 0.34 and 0.41 mM , respectively.