Purification and properties of the first-identified, archaeal, ATP-dependent 6-phosphofructokinase, an extremely thermophilic non-allosteric enzyme, from the hyperthermophile Desulfurococcus amylolyticus

摘要

The ATP-dependent 6-phosphofructokinase (ATP-PFK) of the hyperthermophilic archaeon Desulfurococcus amylolyticus was purified 1500-fold to homogeneity. The enzyme had an apparent molecular mass of 140 kDa and was composed of a single type of subunit of 33 kDa suggesting a homotetrameric (alpha(4)) structure. The N-terminal amino acid sequence did not show significant similarity to ATP-PFKs isolated from eubacteria and eukarya. Kinetic constants of the enzyme were determined for both reaction directions at pH 6 and at 85 degrees C. Rate dependence on all substrates followed Michaelis-Menten kinetics. The apparent K(m)s for ATP and fructose 6-phosphate (forward reaction) were 0.28 and 1.17 mM, respectively; the apparent V-max was about 41 U/mg. ATP could not be replaced by pyrophosphate (PP,) or ADP as phosphoryl donor, thus defining the enzyme as an ATP-dependent PFK. In addition to ATP (100%), the enzyme accepted GTP (97%), ITP (130%), UTP (84%), CTP (55%) and, less effectively, acetyl phosphate (13%) as phosphoryl donors. Enzyme activity was not allosterically regulated by classical effecters of ATP-PFKs such as ADP, AMP, and phosphoenolpyruvate or citrate. The enzyme also catalysed in vitro the reverse reaction with an apparent K-m for fructose-l,6-bisphosphate and ADP of 16.7 and 0.5 mM, respectively, and an apparent V-max of about 4.5 U/mg. Divalent cations were required for maximal activity; Mg2+, which was most effective, could be replaced partially by Ni2+, Mn2+ or Co2+. The enzyme had a temperature optimum of 90 degrees C and showed a significant thermostability up to 100 degrees C, which is in accordance with its physiological function under hyperthermophilic conditions. This is the first description of an ATP-dependent PFK from the domain of archaea, characterized as an extremely thermophilic, non-allosteric enzyme. [References: 25]