Scott H. Freeburg '16 and Rachel Schafer '18


I. Introduction

Glycolysis is an oxygen-independent metabolic pathway that carries out the catalysis of glucose into two molecules of pyruvate, while also generating two molecules of ATP. Phosphofructokinase (PFK) carries out the first committed step of glycolysis, the conversion of fructose-6-phosphate to fructose-1,6-bisphosphate. In this reaction, PFK phosphorylates fructose-6-phosphate following cleavage of a phosphate group from ATP.

PFK activity is subject to complex control by a cohort of small-molecule regulators. The most active form of PFK, held in the "relaxed" state, is induced by ADP binding at an allosteric site, triggering a conformational change that elevates the affinity of the active site for fructose-6-phosphate. In contrast, phosphoenolpyruvate can bind to the same allosteric site, eliciting a "closed" conformation with diminished affinity for fructose-6-phosphate. Together, these allosteric regulators exert tight control over PFK activity.  

II. General Structure

Phosphofructokinase is a homotetramer of 34 kDA. Each consists of 319 amino acids. Momomeric units of PFK are comprised of . Domain 1 is comprised of six and seven . While the central sheets of domain 1 are parallel, the outer sheets run in the opposite direction. Domain 2 consists of six and four . The central two beta sheets into the the substrate binding cavity that is occupied by fructose-6-phosphate when PFK is in its liganded state.

Tetramerization is coordinated by interactions, which are depicted in a dimer for clarity. For example, dimers are stabilized by interactions between Ile147 and Ile262, which are the principal interactions that enable tetramerization between PFK monomers are with adjacent monomers. However, Asp59 and Arg63 of opposite subunits to stabilize the tetramer. 

III. Formation of Fructose 1,6-Bisphosphate

The two domains of monomeric PFK form an active site that is occupied by fructose-6-phosphate. F6P binding is largely coordinated by interactions with residues within domain 2. F6P is stabilized by of domain 2. In addition, the ring of F6P is held by . ATP also binds to the active site; however, this substrate interacts with residues in domain 1. ATP is stabilized by multiple interactions, including hydrogen bonds with its amino group and Gln 107, hydrophobic interactions with Lys 77, and hydrogen bonds between the 2’ and 3’ hydroxyls of the sugar and Cys 73. ATP is believed to interact with Mg 2+ ions, but the details of this relationship are not yet known. This positioning of the molecule allows for the hydroxyl moiety of F6P to perform a nucleophilic attack of the gamma phosphate of ATP. This phosphorylation results in the formation of fructose-1,6-bisphosphate and ADP.

IV. Allosteric Regulation

The allosteric binding site is found between two of PFK’s monomers. At this site, both ADP and phosphoenolpyruvate (PEP) may bind to regulate PFK activity. ADP causes the protein to be in the “relaxed” state, which increases the affinity of the active site for its substrates. The alpha and beta phosphates of ADP coordinate binding at the allosteric site of PFK. projects into the allosteric binding cavity, interacting with negatively charged oxygen atoms of the beta phosphate group. Meanwhile, oxygens of the alpha phosphate group of ADP interact with as well as . Additional stabilization of ADP is conferred by an that is nestled between the phosphate groups of ADP. these interactions stablize the negative charge of ADP phosphate groups. Other regions of ADP are held by interactions with the adjacent PFK monomer (not depicted)

Inhibition by phosphoenolpyruvate (PEP) offers an additional opportunity for allosteric regulatory control. PEP inhibits PFK activity by eliciting a conformational change that diminishes affinity of the active site for F6P and ATP Since PEP syntheisis is downstream of F6P formation in glycolyis, this feedback inhibition enables fine-tuned regulation of glycolysis. PEP binding is stabilized by interactions with its carboxylate acid and phosphate groups. Interestingly,the carboxylate The negatively charged carboxyl region is held by the main-chain acid is held not by the R group but by the main-chain amide of . Similar to ADP binding, also plays an important role in coordinating with the phosphate group of PEP in the inhibited state of PFK.

V. References

"ATP: Adenosine Triphosphate - Boundless Open Textbook." Boundless. N.p., n.d. Web. 12 Dec. 2015.

Evans, P. R., Farrants, G. W., Hudson, P. J. 1981. Phosphofructokinase: structure and control Phil. Trans. R. Soc. Lond. B 293: 53-62.

"Glycolysis." UC Davis ChemWiki, 01 Oct. 2013. Web. 12 Dec. 2015.

"Glycolysis: Step 3." Karbohidrat Ii Glikolisis. SlideShare, 22 Oct. 2014. Web. 12 Dec. 2015.

Mosser, Rockann, Manchi, C. M. Reddy, Bruning, John B., Sacchettini, James C. 2012. Structure of the Apo Form of Bacillus stearothermophilus Phosphofructokinase. Biochemistry 51:769-775.

Schirmer, Tilman, Evans, Philip R. 1990. Structural basis of the allosteric behaviour of phosphofructokinase. Nature 343:140-145.

Back to Top