Propogation of the Allosteric Signal in Bacillus Stearothermophilus Phosphofructokinase Examined by Methyl-TROSY NMR

Phosphofructokinase from Bacillus stearothermophilus (BsPFK) is a 136 kDa homotetromeric enzyme. Binding of the substrate, fructose 6-phospate (F6P), is allosterically regulated by the K-type inhibitor phospho(enol)pyruvate (PEP). The allosteric coupling between substrate and inhibitor is quantified by an equilibrium constant of the reaction between F6P-bound and PEP-bound complexes forming the apo form and ternary complex. Methyl-TROSY NMR was employed to gain structural information on BsPFK in all four states of ligation that are relevant to the allosteric coupling. BsPFK was uniformly labeled with 15N and 2H and specifically labeled with δ-[13CH3]-isoleucine using an isotopically-labeled α-keto butyric acid precursor. Methyl-TROSY experiments were conducted on BsPFK in 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. These spectral maps reveal several cross peaks with chemical shifts unique to each state of ligation indicating unique structures for each enzyme form. Apo BsPFK and F6P-bound BsPFK spectra exhibit only small differences compared to the PEP-bound BsPFK spectrum, which contains numerous dissimilar peaks. Additional distinct peaks, not seen in any other spectra, are present in the spectrum of the ternary complex. Assignments of all 30 isoleucines were determined through 3D HMQC-NOESY experiments with [U-15N,2H];Ileδ1-[13CH3]-BsPFK and complementary HNCA and HNCOCA experiments with [U-15N,2H,13C]-BsPFK. The assignments allowed for the mapping of peaks representing isoleucine residues onto the crystal structure. This analysis has allowed specific regions of the enzyme involved in the binding of allosteric ligands and the propagation of the allosteric effect to be identified. Funding: NIH-GM33216, NIH-CBI, Welch-A1543.