Scott H. Freeburg '16 and Rachel Schafer '18
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
. While the central sheets of domain 1 are parallel, the outer sheets
run in the opposite direction. Domain 2 consists of six
. 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
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
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.
"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
"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
Schirmer, Tilman, Evans,
Philip R. 1990. Structural basis of the allosteric
behaviour of phosphofructokinase. Nature
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