Effect of Hydrostatic Pressure on Allosteric Couplings in a Tryptophan-Shifted Variant of Phosphofructokinase from Bacillus Stearothermophilus

Abstract

Phosphofructokinase from Bacillus stearothermophilus is an allosteric, homotetrameric enzyme containing one tryptophan per subunit. Unlike its homolog from E. coli, the fluorescence of the native tryptophan is unresponsive to ligand binding. This study utilizes a tryptophan-shifted mutant (W179F/F240W) that is functionally similar to wild-type, however a decrease in fluorescence intensity of about 6.5% is associated with substrate, fructose 6-phosphate (Fru-6-P), binding and a decrease of approximately 20% is associated with inhibitor, phosphoenol pyruvate (PEP), binding. Dissociation constants equal 1.9±0.3 μM for Fru-6-P and 107±13 μM for PEP. This dissociation constant for PEP agrees with that determined kinetically (128±5 μM). Due to MgATP antagonism, the dissociation constant for Fru-6-P in the absence of MgATP is lower than at saturating MgATP (36±1 μM) as determined by steady-state kinetic assays. The coupling between PEP and Fru-6-P increases with temperature and 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 relationship is opposite that for the allosteric response in EcPFK, where the sign is established by the enthalpy component. Fluorescence intensity of the BsPFK variant increases by about 15% linearly as a function of pressure from 0 to 2 kbar. Pressure induced changes to the fluorescence intensity in this pressure range are completely reversible and the enzyme retains 100% of its activity. This variant is being used to determine how the thermodynamic components of the coupling free energy change as a function of pressure with the goal of better understanding the thermodynamic forces involved in the allosteric effect. Initial results indicate that the coupling in BsPFK is relatively pressure insensitive at 25°C. Funding: NIH-GM33261, NIH-CBI, Welch-A1543.