Purification and characterization of an ADP-dependent phosphofructokinase from Thermococcus zilligii.

The ADP-dependent phosphofructokinase (PFK) from Thermococcus zilligii has been purified 950 fold; it had a specific activity of 190 Umg(-1). The enzyme required Mg2+ ions for optimal activity and was specific for ADP. The forward reaction kinetics were hyperbolic for both cosubstrates (pH optimum of 6.4), and the apparent Km values for ADP and fructose-6-phosphate were 0.6mM (apparent Vmax of 243Umg(-1)) and 1.47mM (apparent Vmax of 197Umg(-1)), respectively. Significantly, the enzyme is indicated to be nonallosteric but was slightly activated by some monovalent cations including Na+ and K+. The protein had a subunit size of 42.2kDa and an estimated native molecular weight of 66kDa (gel filtration). Maximal reaction rates for the reverse reaction were attained at pH 7.5-8.0, and the apparent Km values for fructose-1,6-bisphosphate and AMP were 0.56mM (apparent Vmax of 2.9Umg(-1) and 12.5mM, respectively. The biochemical characteristics of this unique ADP-dependent enzymatic activity are compared to ATP and pyrophosphate-dependent phosphofructokinases.