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

Abstract

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 PEPbound 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 15 N and 2 H and specifically labeled with δ-[ 13 CH 3 ]-isoleucine utilizing an isotopically labeled α-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 13 C and 1 H. Assignments for 17 isoleucines were determined through three-dimensional HMQC-NOESY experiments with [U-15 N, 2 H];Ileδ1-[ 13 CH 3 ]-BsPFK and complementary HNCA and HNCOCA experiments with [U-2 H, 15 N, 13 C]-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.

What is allosteric regulation? Exploring the exceptions that prove the rule!

“…Instead, the two binding events must have an overlapping influence on the same area of the protein ( Fig. 1 C ) ( 4 , 74 ), with the active site as only one possible location for that overlapping influence.…”

“…( 4 ) for further discussion). In fact, there are very few systems for which equivalent biophysical data types have been generated to allow comparisons among all four complexes ( 46 , 47 , 48 , 49 , 52 , 74 , 75 , 76 ).…”

“…Also, binding of a ligand in one binding site does not necessitate a change directly in a second ligand binding site when that ligand is not bound. Instead, some area of the protein must be responsive ( i.e ., energetically coupled) to the binding of both types of ligands ( 4 , 74 , 149 ). Allosteric site (effector site) The binding site on the protein to which the allosteric effector binds.…”

Rheostatic contributions to protein stability can obscure a position's functional role

“…Some clues may be obtained from the known differences between ZmPYK and hLPYK: In addition to their different thermal stabilities (Meneely et al, 2023), two other notable differences are their oligomeric states (dimeric ZmPYK vs. tetrameric hLPYK) (McFarlane et al, 2019;Meneely et al, 2023) and allosteric regulation (nonallosteric ZmPYK and allosteric hLPYK) (Page et al, 2022;Steiner et al, 1998). Intriguingly, the latter involves both inter-and intra-monomer communication (Fenton et al, 2003;Fenton & Blair, 2002;Fenton & Reinhart, 2009;Reinhart, 1983Reinhart, , 1988Reinhart, , 2004Tang & Fenton, 2017;Whitaker et al, 2019;Wu et al, 2019), which may also be important for rheostatic changes on functional parameters (Campitelli et al, 2021(Campitelli et al, , 2023.…”