Examination of MgATP binding in a tryptophan-shift mutant of phosphofructokinase from Bacillus stearothermophilus

A tryptophan-shift variant of Bacillus stearothermophilus phosphofructokinase (BsPFK), W179F/F76W, was constructed to evaluate the binding and allosteric characteristics associated with MgATP. W179F/F76W BsPFK has a specific activity of 77 ± 1 U/mg at pH 7 and 25 °C, which is a 35% decrease compared to the wild-type enzyme. The Km for MgATP increases from 43 ± 3 μM for wild-type BsPFK to 160 ± 7 μM in the variant. Binding and allosteric interaction between Fru-6-P and PEP for the variant are similar to those of the wild-type enzyme. W179F/F76W BsPFK has distinct fluorescence properties relative to wild-type or other tryptophan-shifted mutants of BsPFK. The binding of MgATP produces an 80% decrease in the fluorescence intensity while MgADP causes a 70% decrease. Capitalizing on these fluorescence changes, dissociation constants of 30 ± 1 μM and 0.53 ± 0.02 mM were measured for MgATP and MgADP, respectively. In addition, PEP was shown to enhance MgATP binding by 2.6-fold.