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(-l). 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 K-m values for ADP and fructose-6-phosphate were 0.6 mM (apparent V-max of 243 Umg(-l)) and 1.47 mM (apparent V-max of 197 Umg(-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 42.2 kDa and an estimated native molecular weight of 66 kDa (gel filtration). Maximal reaction rates for the reverse reaction were attained at pH 7.5-8.0, and the apparent K-m values for fructose-1,6-bisphosphate and AMP were 0.56 mM (apparent V-max of 2.9 Umg(-1)) and 12.5 mM, respectively. The biochemical characteristics of this unique ADP-dependent enzymatic activity are compared to ATP and pyrophosphate-dependent phosphofructokinases.