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

J Biol Chem. 1999 Jul 23;274(30):21023-8. doi: 10.1074/jbc.274.30.21023.


Pyrococcus furiosus uses a modified Embden-Meyerhof pathway involving two ADP-dependent kinases. Using the N-terminal amino acid sequence of the previously purified ADP-dependent glucokinase, the corresponding gene as well as a related open reading frame were detected in the genome of P. furiosus. Both genes were successfully cloned and expressed in Escherichia coli, yielding highly thermoactive ADP-dependent glucokinase and phosphofructokinase. The deduced amino acid sequences of both kinases were 21.1% identical but did 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 activity of 88 units/mg at 50 degrees C and a pH optimum of 6.5. As phosphoryl group 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, respectively. The phosphofructokinase did not catalyze the reverse reaction, nor was it regulated by any of the known allosteric modulators of ATP-dependent phosphofructokinases. ATP and AMP were identified as competitive inhibitors of the phosphofructokinase, raising the K(m) for ADP to 0.34 and 0.41 mM, respectively.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Diphosphate / metabolism
  • Amino Acid Sequence
  • Cloning, Molecular
  • Genes, Archaeal
  • Kinetics
  • Molecular Sequence Data
  • Phosphofructokinase-1 / genetics*
  • Phosphofructokinase-1 / metabolism*
  • Pyrococcus furiosus / enzymology*
  • Sequence Alignment
  • Substrate Specificity


  • Adenosine Diphosphate
  • Phosphofructokinase-1

Associated data

  • GENBANK/AF127909
  • GENBANK/AF127910