Phosphofructokinase (Bacillus stearothermophilus)
Created by Yun Jae Jo
Phosphofructokinase(pdb id=4pfk) from Bacillus stearothermophilus is an enzyme that catalyzes the phosphorylation of fructose-6-phosphate (F6P) by adenosine triphosphate (ATP). The physiological form of phosphofructokinase is a tetramer with four identical subunits (1). Phosphofructokinase consists of alpha helices, beta sheets, turns, and random coils. Both domains for each subunit have an alpha-beta-alpha sandwich structure (2). The larger domain consists of seven strands of beta sheet. The central five beta strands are parallel and the outer two beta strands are anti-parallel to the others (1). The smaller domain consists of four strands of beta sheet, all of which are parallel strands (1). The active site is formed between the cleft of the two domains, where both beta sheets are directed (1).
The active site of phosphofructokinase lies between two domains of one subunit, and the binding site for F6P is located between the subunits (1). The binding of F6P is essential to the regulated step of glycolysis, where the F6P is phosphorylated at the C-1 position to yield F1,6P2 (7). When the reaction is coupled to ATP hydrolysis by phosphofructokinase, the reaction becomes exergonic, with ∆ G of -14.2 kJ/mol (7). The enzyme activity is high at pH of 7 and at 37 oC. (7). Through the phosphofructokinase reaction, the cell commits to metabolizing glucose (7). The 6-phosphate group of F6P is bound by His-249 and Arg-252 from its small domain and by Arg-162 and Arg-243 from the larger domain of the other subunit (1). The active R state is favored when the activator F6P is bound (1). The higher affinity for F6P distinguishes the active R state of the enzyme from the less active T state (pdb id=6PFK (2))(1). In the active R state, F6P and adenosine triphosphate (ATP) are bridged by Arg-72 (2). The less active T state is favored with the absence of the activator ligand F6P and the binding of the inhibitor phosphoenolpyruvate (PEP). The conformational change of the enzyme is determined by the binding of Arg-162 and Arg-243 to the ligand F6P (1). The change in dimer-dimer interface of the tetramer was detected which confirmed the conformational change between R and T state (2). Arg-72 and Glu-241 form a new salt bridge between the dimers and provide stability for the T state (2). In addition, Arg 252 is central to a network of hydrogen bonds, linking F6P to Asn-12 from the same subunit, and His-160 and Thr-156 from the other subunit (1).
The binding site of ATP is located between an alpha helix (residues 102-108) and a surface loop between helices (residues 72-77) (1). The binding of ATP is functionally critical to the phosphorylation of F6P. The carbonyl of Cys-73 and the main chain nitrogen are also hydrogen bonded to two ribose hydroxyls of ATP (1). Gln-107 is hydrogen-bonded to the N6 amino group of the ATP, and the side chain of Lys-77 has hydrophobic contact with the other side of adenine (1).
ADP is bound at the effector site between two subunits along the x dyad, where the diphosphate group and Mg2+ ion are buried in the cleft with the adenine group on the surface (1). ADP serves as the allosteric activator of the control step in glycolysis (7). The ADP and Mg2+ complex increases the enzyme's affinity for F6P. An inorganic phosphate binds in the place of the beta phosphate position of ADP in the native crystal, along with the phosphate group of PEP, which inhibits the enzyme by decreasing its affinity for F6P (1). Conformational changes in the binding site are caused by Mg (1). Residues 212-215 of the main chain shifts downwards over the ADP (1). Furthermore, the ribose of ADP binds with the magnesium ion as His-215 moves out of the path (1).
The tryptophan-shift mutant (pdb id=1MTO) of phosphofructokinase from Bacillus stearothermophilus was compared to the wild type enzyme because of their primary structure similarities (3). The mutant of PFK was chosen because it was the only protein with known structural coordinates among the proteins with a non-zero E value of 4e-180. The crystal structure of the mutant, designated W179Y/Y164W, did not show dramatic alterations in the tertiary structure of the enzyme (3). However, the mutant enzyme underwent a reversible dissociation at the active site interface when PEP was bound to the allosteric site (3). It was suggested that the allosteric communication was affected by the quaternary structural changes due to the tryptophan shift (3). The mutant enzyme showed kinetic and thermodynamic properties which were similar to the wild type enzyme with several changes (3). The kcat showed a twofold decrease, and K0.5 showed a threefold decrease for the substrate F6P (3). The decrease in the mutant enzyme activity suggested an alteration in the structure of the binding site (3). The dissociation constant for PEP showed a twofold increase, but it was determined that PEP was still an effective inhibitor (3). However, attempts of the crystallization of the mutant enzyme with PEP have been unsuccesful (3).
The phosphofructokinase of Lactobacillus bulgaricus (4) (LbPFK, pdb id=1ZXX) was compared to the wild type enzyme (BsPFK) through the Dali server (5). The tertiary structural similarities had a root mean squared deviation of 0.8 and a Z score of 48.4 (5). In comparison of LbPFK and BsPFK, the folds and substrate binding site were conserved (4). However, the LbPFK showed a decrease in its binding affinity toward its allosteric ligands, PEP and MgADP (4). Unlike the typical allosteric behavior of the wild type enzyme (based on extensive communication between the four subunits) (6), the decreased binding affinity is likely due to the change in the residues at the allosteric site (4). Unlike the wild type enzyme, BsPFK, both allosteric ligands show an inhibitory effect in LbPFK with no clear answer (4). The LbPFK showed an insignificant binding affinity toward allosteric ligands at a high pH, which conveyed that LbPFK was a nonallosteric enzyme at a high pH (4).