ADP-dependent 6-phosphofructokinase, an extremely thermophilic, non-allosteric enzyme from the hyperthermophilic, sulfate-reducing archaeon Archaeoglobus fulgidus strain 7324

Abstract

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°C and showed significant thermostability up to 95°C. Kinetic constants were determined for both reaction directions at pH 6.6 and 80°C. Rate dependence for all substrates followed Michaelis Menten kinetics. The apparent K _m for ADP and fructose-6-phosphate (forward reaction) was 0.6 mM and 2.2 mM, respectively; the apparent V _max was 1,200 U/mg. ADP-PFK catalyzed in vitro the reverse reaction, with apparent K _m for fructose-1,6-bisophosphate and AMP of 5.7 and 1.4 mM, respectively, and a V _max 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.