Redefining the Role of the Quaternary Shift in the Allosteric Inhibition of Bacillus Stearothermophilus Phosphofructokinase

Abstract

Bacillus stearothermophilus PFK (BsPFK) is a homotetramer that is allosterically inhibited by phosphoenolpyruvate (PEP), which binds along one dimer-dimer interface. The substrate, fructose-6-phosphate (Fru-6-P), binds along the other dimer-dimer interface. The inhibitor-bound structure compared to the substrate-bound structure of wild-type BsPFK exhibits a 7° rotation about the substrate binding interface, termed the quaternary shift. Evans, et. al. proposed that the quaternary shift is the mechanism for allosteric inhibition for BsPFK. However, the main role of the quaternary shift may be in ligand binding and not allosteric inhibition. The variant D12A BsPFK shows a 100-fold increase in the binding affinity for PEP, a 50-fold decrease in the binding affinity for Fru-6-P, and a coupling comparable to wild-type. Crystal structures of apo and PEP bound forms of D12A BsPFK both indicate a shifted structure similar to the inhibitor-bound structure of wild-type. Remarkably, D12 does not directly bind to either substrate or inhibitor, and is located along the substrate binding interface. A conserved hydrogen bond between D12 and T156 takes place across the substrate binding interface in the substrate-bound form of BsPFK. The variant T156A BsPFK, when compared to wild-type, shows a 30-fold increase in PEP binding affinity, a 17-fold decrease in Fru-6-P binding affinity, and an estimated coupling that is at least wild-type coupling. In addition, T156A BsPFK crystal structure exhibits a shifted structure similar to D12A BsPFK and the inhibitor-bound structure of wild-type. PEP still inhibits these variants of BsPFK despite the fact that the enzymes are in the quaternary shifted position prior to PEP binding. Therefore the quaternary shift of BsPFK primarily perturbs ligand binding but does not directly contribute to heterotropic allosteric inhibition. Supported by NIH Grant GM33216 and Welch Foundation Grant A1548.