The ATP-dependent 6-phosphofructokinase (ATP-PFK) of the hyperthermophilic bacterium Thermotoga maritima was 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 75degreesC. 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 for fructose 1,6-bisphosphate and ADP of 7.6 mM and 1.4 mM, respectively, and an apparent l,V-max of about 13 U/mg. Divalent cations were required for maximal activity; Mg2+, which was most effective, could partially be replaced by Mn2+ and Fe2+. 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 93degreesC and showed a significant thermostability up to 100degreesC. 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.
