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


  • Phosphofructokinase from Bacillus stearothermophilus (BsPFK) is a 136 kDa homotetromeric enzyme. Binding of the substrate, fructose 6-phosphate (Fru-6-P), is allosterically regulated by the K-type inhibitor phosphoenolpyruvate (PEP). The allosteric coupling between the 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-transverse relaxation-optimized spectroscopy (Me-TROSY) nuclear magnetic resonance was employed to gain structural information about BsPFK in all four states of ligation relevant to the allosteric coupling. BsPFK was uniformly labeled with 15N and 2H and specifically labeled with -[13CH3]-isoleucine utilizing an isotopically labeled -keto acid isoleucine precursor. Me-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 17 isoleucines were determined through three-dimensional HMQC-NOESY experiments with [U-15N,2H];Ile1-[13CH3]-BsPFK and complementary HNCA and HNCOCA experiments with [U-2H,15N,13C]-BsPFK. The assignments allowed for the mapping of resonances representing isoleucine residues to a previously determined X-ray crystallography structure. This analysis, performed for all four states of ligation, has allowed specific regions of the enzyme influenced by the binding of allosteric ligands and those involved in the propagation of the allosteric effect to be identified and distinguished from one another.

published proceedings

  • Biochemistry

author list (cited authors)

  • Whitaker, A. M., Naik, M. T., Mosser, R. E., & Reinhart, G. D.

citation count

  • 1

complete list of authors

  • Whitaker, Amy M||Naik, Mandar T||Mosser, Rockann E||Reinhart, Gregory D

publication date

  • January 2019