ATP-dependent 6-phosphofructokinase from the hyperthermophilic bacterium Thermotoga maritima: characterization of an extremely thermophilic, allosterically regulated enzyme.
The ATP-dependent 6-phosphofructokinase (ATP-PFK) of the hyperthermophilic bacterium Thermotoga maritimawas purified 730-fold to homogeneity. The enzyme is a 140-kDa homotetramer composed of 34 kDa subunits. Kinetic constants were determined for all substrates in both reaction directions at pH 7 and at 75 degrees C. Rate dependence (forward reaction) on fructose 6-phosphate (F-6-P) showed sigmoidal kinetics with a half-maximal saturation constant ( S(0.5)) of 0.7 mM and a Hill coefficient of 2.2. The apparent K(m) for ATP was 0.2 mM and the apparent V(max) value was about 360 U/mg. The enzyme also catalyzed in vitro the reverse reaction with an apparent K(m) for fructose 1,6-bisphosphate and ADP of 7.6 mM and 1.4 mM, respectively, and an apparent V(max) of about 13 U/mg. Divalent cations were required for maximal activity; Mg(2+), which was most effective, could partially be replaced by Mn(2+) and Fe(2+). Enzyme activity was allosterically regulated by classical effectors of ATP-PFKs of Eukarya and Bacteria; it was activated by ADP and inhibited by PEP. The enzyme had a temperature optimum of 93 degrees C and showed a significant thermostability up to 100 degrees C. Using the N-terminal amino acid sequence of the subunit, the pfk gene coding for ATP-PFK was identified and functionally overexpressed in Escherichia coli. The purified recombinant ATP-PFK had identical kinetic and allosteric properties as the native enzyme purified from T. maritima. The deduced amino acid sequence showed high sequence similarity to members of the PFK-A family. In accordance with its allosteric properties, ATP-PFK of T. maritima contained the conserved allosteric effector-binding sites for ADP and PEP.
Hansen
Thomas
T
Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität Kiel, Am Botanischen Garten 1-9, 24118 Kiel, Germany.
Musfeldt
Meike
M
Schönheit
Peter
P
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005947:Glucose / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010732:Phosphofructokinase-1 / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature; D020124:Thermotoga maritima / Q000201:enzymology* / Q000378:metabolism
D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1; D005947:Glucose