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 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.
Laboratory of Microbiology, Department of Biomolecular Sciences, Wageningen Agricultural University, Hesselink van Suchtelenweg 4, 6703 CT Wageningen, The Netherlands. Judith.Tuininga@algemeen.micr.wau.nl
J E
JE
Tuininga
C H
CH
Verhees
J
J
van der Oost
S W
SW
Kengen
A J
AJ
Stams
W M
WM
de Vos
D000244:Adenosine Diphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D019766:Genes, Archaeal; D007700:Kinetics; D008969:Molecular Sequence Data; D010732:Phosphofructokinase-1 / Q000235:genetics* / Q000378:metabolism*; D020027:Pyrococcus furiosus / Q000201:enzymology*; D016415:Sequence Alignment; D013379:Substrate Specificity
D000244:Adenosine Diphosphate; D010732:Phosphofructokinase-1