Fluorescent Binding Studies of Phosphofructokinase from Bacillus Stearothermophilus Using a Tryptophan-Shifted Mutant

Abstract

Phosphofructokinase from Bacillus stearothermophilus (BsPFK) is an allosteric, homotetrameric enzyme containing one tryptophan per subunit. Unlike the homologous PFK from E. coli (EcPFK), the fluorescence of the native tryptophan is unresponsive to ligand binding. This study utilizes a tryptophan-shifted mutant, in which W179 has been mutated to phenylalanine, and F240 has been mutated to tryptophan. The variant is functionally similar to wild-type, however a decrease in the fluorescence of 6.5% is associated with substrate fructose 6-phosphate (Fru-6-P) binding and a decrease of 16% is associated with inhibitor PEP binding. Dissociation constants of 1.9±0.3μM for Fru-6-P and 107±13μM for PEP were thereby determined. This dissociation constant for PEP is in good agreement with that determined by kinetic assays (128±5μM). Due to the absence of known ATP antagonism, the dissociation constant for Fru-6-P in the absence of MgATP is lower than with steady-state kinetic assays determined at saturating MgATP (36±1μM). The coupling between PEP and Fru-6-P has also been determined and increases with temperature as observed with steady-state kinetic assays using wild-type BsPFK. Like wild-type PFK, the coupling free energy results from compensating enthalpy and entropy components. The sign of the coupling free energy is opposite that of the enthalpy and is therefore determined by the larger absolute value of the entropy term. This is opposite the thermodynamic basis of the allosteric response in EcPFK, where the sign is established by the enthalpy component. The functional and thermodynamic similarity of the mutant enzyme to wild-type, together with the added ability to follow ligand binding with fluorescence, make it a good candidate for further studies probing a deeper understanding of the thermodynamics involved in PFK allosterism. Funding provided by the following: NIH-GM33216, NIH-CBI, and the Welch Foundation.