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

abstract

• 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.

authors

• Reinhart, Gregory

published proceedings

• Arch Biochem Biophys

author list (cited authors)

• Riley-Lovingshimer, M. R., & Reinhart, G. D.

citation count

• 4

complete list of authors

• Riley-Lovingshimer, Michelle R||Reinhart, Gregory D

publication date

• January 2005

publisher

• Elsevier  Publisher

published in

• Archives of Biochemistry and Biophysics  Journal

keywords

• Adenosine Diphosphate
• Adenosine Triphosphate
• Allosteric Site
• Base Sequence
• Binding Sites
• Fructosephosphates
• Genetic Variation
• Geobacillus Stearothermophilus
• Mutation
• Phosphoenolpyruvate
• Phosphofructokinases
• Spectrometry, Fluorescence
• Tryptophan

Digital Object Identifier (DOI)

• 10.1016/j.abb.2005.01.020

start page

• 178

end page

• 186

volume

• 436

issue

• 1

URL

• http%3A%2F%2Fdx.doi.org%2F10.1016%2Fj.abb.2005.01.020