ADP-dependent 6-phosphofructokinase, an extremely thermophilic, non-allosteric enzyme from the hyperthermophilic, sulfate-reducing archaeon Archaeoglobus fulgidus strain 7324.
The hyperthermophilic, sulfate-reducing archaeon Archaeoglobus fulgidus strain 7324 has been shown to degrade starch via glucose using a modified Embden-Meyerhof pathway. In this pathway phosphorylation of fructose-6-phosphate to fructose-1,6 bisphosphate is catalyzed by an ADP-dependent 6-phosphofructokinase (ADP-PFK), which was purified 1,800-fold to homogeneity. The enzyme is composed of 50 kDa subunits and is eluted from gel filtration as both a homotetramer and a homodimer. It had a temperature optimum at 85 degrees C and showed significant thermostability up to 95 degrees C. Kinetic constants were determined for both reaction directions at pH 6.6 and 80 degrees C. Rate dependence for all substrates followed Michaelis Menten kinetics. The apparent Km for ADP and fructose-6-phosphate (forward reaction) was 0.6 mM and 2.2 mM, respectively; the apparent Vmax was 1,200 U/mg. ADP-PFK catalyzed in vitro the reverse reaction, with apparent Km for fructose-1,6-bisophosphate and AMP of 5.7 and 1.4 mM, respectively, and a Vmax value of 85 U/mg. The enzyme did not use ATP, PPi, or acetyl phosphate as phosphoryl donor and was highly specific for fructose-6-phosphate as substrate. The A. fulgidus ADP-PFK did not phosphorylate glucose and thus differs from the bifunctional ADP-PFK/GLK from Methanococcus jannaschii. Divalent cations were required for catalytic activity; Mg2+, which was most effective, could be partially replaced by Mn2+, Ni2+, and Co2+. Enzyme activity was not allosterically regulated by classical effectors of bacterial and eukaryal ATP-PFKs, such as ADP, AMP, phosphoenolpyruvate, or citrate. N-terminal amino acid sequence showed high similarity to known ADP-PFKs. In the genome of Archaeoglobus fulgidus strain VC 16, which is closely related to strain 7324, no homologous gene for ADP-PFK could be identified.
Hansen
Thomas
T
Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität Kiel, Am Botanischen Garten 1-9, 24118, Kiel, Germany.
Schönheit
Peter
P
D000595:Amino Acid Sequence; D019611:Archaeoglobus fulgidus / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D021122:Protein Subunits; D017386:Sequence Homology, Amino Acid
D021122:Protein Subunits; D017853:Phosphotransferases (Alcohol Group Acceptor); C442129:ADP D-fructose-6-phosphate 1-phosphotransferase