Propagation of the Allosteric Signal in Phosphofructokinase from Bacillus stearothermophilus Examined by Methyl-Transverse Relaxation-Optimized Spectroscopy Nuclear Magnetic Resonance

Phosphofructokinase from Bacillus stearothermophilus (BsPFK) is a 136 kDa homotetromeric enzyme. Binding of the substrate, fructose-6-phospate (Fru-6-P), is allosterically regulated by K-type inhibitor phospho-(enol)pyruvate (PEP). The allosteric coupling between substrate and inhibitor is quantified by a standard coupling free energy that defines an equilibrium with the Fru-6-P bound and PEP bound complexes on one side and the apo form and ternary complex on the other. Methyl-TROSY NMR was employed in order to gain structural information on BsPFK in all four states of ligation relevant to the allosteric coupling. BsPFK was uniformly 15N and 2H labeled and specifically labeled with δ-[13CH3]-isoleucine utilizing an isotopically labeled α-keto acid isoleucine precursor. Methyl-TROSY experiments were conducted on all four ligation states and all 30 isoleucines, which are well dispersed throughout each subunit of the enzyme, are well resolved in chemical shift correlation maps of 13C and 1H. Assignments for ...