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. 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 microM 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.
Riley-Lovingshimer
Michelle R
MR
Department of Biochemistry and Biophysics, Texas A&M University and the Texas Agricultural Experiment Station, 2128 TAMU, College Station, TX 77843-2128, USA.
Reinhart
Gregory D
GD
D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000495:Allosteric Site / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D005636:Fructosephosphates / Q000737:chemistry; D014644:Genetic Variation; D001411:Geobacillus stearothermophilus / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010728:Phosphoenolpyruvate / Q000494:pharmacology; D025221:Phosphofructokinases / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000235:genetics*
D005636:Fructosephosphates; D000244:Adenosine Diphosphate; C027618:fructose-6-phosphate; D010728:Phosphoenolpyruvate; D014364:Tryptophan; D000255:Adenosine Triphosphate; D025221:Phosphofructokinases