Redefining the role of the quaternary shift in Bacillus stearothermophilus phosphofructokinase.

Phosphofructokinase-1 (PFK)1 is a highly regulated enzyme that catalyzes the first committed step of glycolysis in prokaryotes. It is one of two non-homologous gene products that catalyze the transfer of the γ-phosphate of MgATP to fructose-6-phosphate (Fru-6-P) producing fructose-1, 6-bisphosphate and MgADP. PFK from the moderate thermophile, Bacillus stearothermophilus (Bs), is a homotetramer of 34 KDa per subunit that forms a dimer of dimers creating two unique subunit-subunit interfaces. One subunit-subunit interface contains four identical binding sites for Fru-6-P per homotetramer. The other subunit-subunit interface contains the four identical binding sites for the allosteric ligands per homotetramer. The allosteric binding sites physiologically are capable of binding either MgADP or phosphoenolpyruvate (PEP). For BsPFK, MgADP serves as a very weak activator and PEP is a very strong inhibitor at 25°C. Both PEP and MgADP alter the affinity BsPFK displays for Fru-6-P without changing the enzyme’s maximal activity; therefore, both allosteric ligands are considered to be K-type effectors. The regulation of PFK has been the subject of study for nearly five decades since Blangy et al. first characterized the kinetics of PFK from E. coli and explained the behavior using a 2-state model (1) in which the 2 states undergo a concerted transition in accordance with the model proposed by Monod, Wyman, and Changeux (2). Schirmer and Evans compared the crystal structure of PFK bound to the inhibitor analog phosphoglycolate (PGA) (PDB accession code 6PFK) (3) with that obtained with BsPFK bound to the Fru-6-P and the activator MgADP (PDB accession code 4PFK) (4, 5) and found a substantial conformational difference. The conformational change includes a difference in the quaternary structures of the two enzyme forms. The quaternary structure of the inhibitor bound BsPFK has undergone a 7° rotation about the substrate binding interface, termed the quaternary shift, when compared to the substrate plus activator bound BsPFK. The conformational change also includes the movement of residues E161 and R162 in the active site. When Fru-6-P is bound to the enzyme, the positively charged side chain of R162 interacts with the negatively charged phosphate group of Fru-6-P. In the PGA bound structure, R162 effectively replaces the negatively charged side chain of E161. Given the structural differences seen between the substrate and activator bound and inhibitor bound structures of BsPFK, Schirmer and Evans proposed that these 2 structures corresponded to the 2 states that Blangy et al. had invoked to explain the allosteric behavior of PFK (3). Since 1990, BsPFK often has been used as a textbook example of the structural basis for allosteric regulation (6, 7). A key distinction that should be made when assessing the allosteric action of a ligand, such as the inhibitor PEP, is between the affinity it displays for the enzyme and the effectiveness with which it inhibits once it binds. The latter attribute is quantitatively conveyed by the coupling between the inhibitor and substrate (8). We have previously shown that the repositioning of R162 and E161 has a surprisingly small role to play in the inhibitory coupling between Fru-6-P and PEP (9). The present investigation suggests that the quaternary shift similarly does not appear to be the basis for the coupling between Fru-6-P and PEP. A significant structural perturbation associated with the quaternary shift is the disruption of several inter-subunit hydrogen bonds involving residue D12. Previous work performed by Ortigosa, et al. noted the significance of D12 when constructing certain hybrid forms of BsPFK (10). In that study, a D12A mutation was made in concert with other mutations in order to allow the proper exchange of subunits when making hybrids. The characteristics of the single mutant were never fully investigated. We report herein the properties of D12A that include the X-ray crystal structures of D12A BsPFK with and without PEP bound (PDB accession numbers 4I36 and 4I7E, respectively). Both structures exhibit a quaternary structure similar to that introduced by the binding of PGA to the wild type enzyme. While D12A alters the binding of PEP and Fru-6-P, it has very little effect on the allosteric coupling. In an effort to further understand the role of D12, a residue that is 100% conserved in over 150 prokaryotic PFK-1 sequences, we also have examined the behavior of variants of BsPFK in which those residues with which D12 interacts across the interface have been changed to alanine. Generally these variants also exhibit comparable or greater coupling between Fru-6-P and PEP. The structure of one of these variants, T156A, bound to PEP has also been determined (PDB Accession number 4I4I), and not surprisingly it also exhibits the quaternary shift. It is of note that both D12A and T156A allowed for the first time structures of BsPFK to be solved in which the native inhibitor, PEP, is bound.