Sandbox/Judy Voet/PFK

Phosphofructokinase (PFK)Phosphofructokinase (PFK)

Phosphofructokinase (PFK) is a glycolytic enzyme that catalyzes the transfer of a phosphoryl group from ATP to fructose-6-phosphate (F6P) to yield ADP and fructose-1,6-bisphosphate (FBP). The PFK reaction is strongly exergonic (irreversible) under physiological conditions and hence is one of the glycolytic pathway's rate-determining steps. In most organisms/tissues, PFK is the glycolytic pathway's major flux-regulating enzyme; its activity is controlled by the concentrations of an unusually large number of metabolites including ATP, ADP, AMP, and fructose-2,6-bisphosphate (F2,6P).

spinqualitylabelsall models PFK: R-state Biological tetramer; generated from 4pfk by QPS Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

PFK from B. stearothermophilus is a tetramer of identical 320-residue subunits. It is an allosteric enzyme that is described using the symmetry model of allosterism whereby there is a concerted transition from its high-activity R state to its low-activity T state. The X-ray structures of both R and T states of the enzyme have been reported. The binding of one molecule of its substrate F6P, which binds to the R state enzyme with high affinity but to the T state enzyme with low affinity, causes PFK to take up the R state, which in turn, increases the binding affinity of the enzyme for additional F6P (a homotropic effect). Activators, such as ADP and AMP bind to so-called allosteric sites, binding sites distinct from the active site, where they likewise facilitate the formation of the R state and hence activate the enzyme (a heterotropic effect; ADP, being a product of the PFK reaction, also binds at the enzyme's active site). Similarly, inhibitors such as PEP bind to allosteric sites (which in the case of PFK overlaps the activating allosteric site) where they promote the formation of the T state, thereby inhibiting the enzyme. Two of the active sites of the enzyme are located at the interface of subunits A (light Blue) and D (yellow) and two at the interface of subunits B (green) and C (pink). Two of the allosteric sites are located at the interface of subunits A and B and two at the interface of subunits C and D. Here is a closeup of the of subunit D (Yellow). Note that amino acids from subunit A (light blue) also contribute to the binding of F6P. Also here is a closeup of the of subunit B with contributions from both subunits to the binding of ADP.

This Kinemage exercise consists of two kinemage scenes that illustrate some of the allosterically-induced conformational changes that occur in PFK from Bacillus stearothermophilus.

1. Conformational Changes in a Dimeric Unit of PFK1. Conformational Changes in a Dimeric Unit of PFK

This kinemage shows the two subunits of the tetramer whose interface contains two active sites. (KineMage currently not supported) The first view, 1. PFK dimer, shows the two subunits in their R state conformation as represented by their Ca backbones with Subunit 1 in pinktint and Subunit 2 in pink. Two side chains in each subunit are shown, those of Glu 161 (red) and Arg 162 (cyan), which are important participants in PFK's allosterically facilitated conformational change (see below). An F6P (hotpink) and an ADP (green; "ADP-active") are bound in the active site of each subunit. An additional ADP (yellow; "ADP-allo") is bound in a separate so-called allosteric site of each subunit. The ADPs each have an associated Mg2+, which is represented here by a ball of the same color as the ADP to which it binds.

Click the "ANIMATE" button to switch the dimer between its R and T states. In its T state, Subunit 1 is bluetint and Subunit 2 is skyblue. The side chains of Glu 161 and Arg 162 in both subunits are red and cyan as before (only the Ca and Cb atoms of the Arg 162 side chain in Subunit 1 are observed in the X-ray structure of the T state; those of Subunit 2 are all observed). The T state enzyme binds the inhibitor 2-phosphoglycolate (gold; "PGC"), a nonphysiological analog of the glycolytic intermediate phosphoenolpyruvate (PEP). Note that the binding site of PGC in the T state overlaps the allosteric binding site of ADP in the R state ("ADP-allo") and hence their binding is mutually exclusive. The T state active sites, which do not contain F6P, are marked by "ghost" F6Ps (gray;"F6P site"), which have the same positions as do the F6Ps in the R state enzyme.

The seconbd view, 2. Allo/Act Sites, is a closeup of the upper portion of the first view showing both the active site and the allosteric site in this region. Note that the active site is located at the interface between two subunits and that the allosteric site interacts directly with the active site on the adjacent subunit. Compare the R state and T state conformations by displaying both at once or clicking on "ANIMATE". Can you identify the Mg2+ ion associated with each of the ADPs bound to the enzyme in the R state? Which ADP atoms coordinate these Mg2+ ions?

The phosphate group of PGC binds to the allosteric site in the T state in very nearly the same position that the beta phosphate group of "ADP-allo" binds to the R state allosteric site; both phosphate groups bind to the side chains of the same three Arg residues (not shown).

In the high-activity R state, the positively charged side chain of Arg 162 forms a hydrogen bonded salt bridge with the negatively charged 6-phosphate group of F6P (white dashed lines), an interaction which presumably stabilizes the R state relative to the T state and is therefore in part responsible for F6P's homotropic effect.

2: The Major Conformational Changes in a Subunit of PFK.2: The Major Conformational Changes in a Subunit of PFK.

This KINEMAGE shows those segments near the allosteric site (residues 53-60 are not shown here). As in KINEMAGE 1, the polypeptide is represented by its Ca chain with R state Subunits 1 and 2 in redtint and pink, and T state Subunits 1 and 2 in bluetint and skyblue. (KineMage currently not supported) KINEMAGE 2 comes up in view 1, The Allosteric Site, in the R state showing the phosphate group of F6P (hotpink) bound in the enzyme's active site in a hydrogen bonded salt bridge (dashed white lines) with the side chain of Arg 162 (cyan). An ADP (yellow; "ADP-allo") occupies the adjacent allosteric site. Click once on "ANIMATE" to switch to the T state. This replaces the ADP in the R state allosteric site with the inhibitor and PEP analog PGC (gold). F6P no longer occupies the active site but its position in the R state is indicated by the "ghost" F6P (gray; viewed by clicking on "F6P site").

What happens to the central polypeptide helical segment (residues 149-164) in the R to T transition? What does this do to the relative positions of the negatively charged Glu 161 and the positively charged Arg 162? Click on "F6P site". What influence would the presence of the carboxylate group of Glu 161 have on the phosphate group of F6P were it to bind in the active site? Does this explain, at least in part, why T state PFK has low affinity for F6P? Go to View 2. Closeup, for a closeup of the F6P-sidechain interactions. Center the molecules by choosing "pickcenter" from the "tools" menu and clicking on athe atom you'd like to be in the center. Slide the "zoom" slider to enlarge the view.

Site-Directed MutagenesisSite-Directed Mutagenesis

Three mutant PFKs have been made, R162A, E161A and R162A/E161A . The R162A mutation caused a 30-fold decrease in F6P binding. Surprisingly, the E161A mutation had little effect on the ability of PEP to inhibit F6P binding. A more complicated analysis of the allosteric effects of PFK have therefore been proposed.

Atomic CoordinatesAtomic Coordinates

The atomic coordinates for R state PFK were obtained from 4pfk; those for T state PFK were obtained from Philip Evans, MRC Laboratory of Molecular Biology, Cambridge, U. K. but are now available as 6pfk.