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.
Tuininga
J E
JE
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
Verhees
C H
CH
van der Oost
J
J
Kengen
S W
SW
Stams
A J
AJ
de Vos
W M
WM
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