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 degrees C, which is a 35% decrease compared to the wild-type enzyme. The K(m) for MgATP increases from 43+/-3 microM for wild-type BsPFK to 160+/-7 microM in the variant.

Reversible ligand-induced dissociation of a tryptophan-shift mutant of phosphofructokinase from Bacillus stearothermophilus.

The biophysical properties of a tryptophan-shifted mutant of phosphofructokinase from Bacillus stearothermophilus (BsPFK) have been examined. The mutant, designated W179Y/Y164W, has kinetic and thermodynamic properties similar to the wild-type enzyme. A 2-fold decrease in kcat is observed, and the mutant displays a 3-fold smaller K(0.