Phosphofructokinase (PDB: 3O8O) from Saccharomyces cerevisiae
Created by: Yazan Alshawkani
Phosphofructokinase (PDB: 6pf3O8O)
from Saccharomyces cerevisiae is a
multisubunit allosteric enzyme responsible for catalyzing the primary
regulatory step in glycolysis: the phosphorylation of fructose 6-phosphate to
fructose 1,6-biphosphate by ATP. Glycolysis is the first step of cellular
respiration in which living cells break down glycose into small,
energy-containing ATP molecules. The crystal structures of eukaryotic
phosphofructokinases from Saccharomyces
cerevisiae also represent how successive gene duplications and fusion in
the protein have allowed the evolution of new functionalities.
Phosphofructokinase plays an
important catalytic role in transient glycolysis that enables mitochondrial
fusion and the stimulation of the S phase entry. This protein is necessary to
upregulate glycolysis at this stage of the cell cycle (8). Elevated
temperatures can hasten pH decline until phosphofructokinase loses its
activity. In most organisms, at a pH below 5.5, the phosphofructokinase loses
activity and, hence, arrests the process of glycolysis because of the protein’s
inability to perform its catalytic role. The deficiency of phosphofructokinase
can interfere with the production of muscle glycogen and ultimately lead to
glycogen storage disease that depresses the health of an individual (3). Additionally,
oscillations are distributed in nature and the synchronization of oscillators are
described at the cellular level of the phosphofructokinase in regards to the
process of glycolysis. Specifically, the allosteric regulation of
phosphofructokinase regulates the emergence of glycolytic oscillations, which
determines the rate of the glycolysis occurring (1). Additionally, a glycolytic
enzyme like phosphofructokinase can favor cancer cell proliferation through
nonmetabolic function. It can participate in the cell cycle activation and can
have an antipoptotic effect (6). The phosphofructokinase enzyme also plays a
special steering function for the regulation of intermediary metabolism in the
liver (7). It efficiently prepares glucose for catabolism and energy production
to sufficiently support these liver cells.
The molecular weight of
phosphofructokinase in Saccharomyces
cerevisiae is 169309.36 Da while its isoelectric point is 6.31 (4). In
eukaryotes, PFKs are activated by fructose 2,6-biphosphate, a potent allosteric
regulator that controls the rate of glycolysis, overrides inhibition by ATP, and
makes PFK in higher organisms sensitive to the activities of the hormones
insulin and glucagon. The model of the 12S Saccharomyces cerevisiae PFK (ScPFK) structure consists of 6068 amino acid residues,
97% of the recrystallized protein, in eight polypeptide chains (5). Eight
molecules of the ligand Fructose-6-Phosphate and eight molecules of the ligand fructose-2,6-diphosphate are bound to the enzyme. The Fructose-6-phosphate and
fructose-2,6-diphosphate are also considered the prosthetic group of the
protein because they are required for the enzyme activity of
phosphofructokinase. The associated fructose-6-phosphate ligand lies within the
glycolysis metabolic pathway and is further phosphorylated to
fructose-1,6-biphosphate. The associated fructose-2,6-diphosphate ligand is a
metabolite that allosterically affects the activity of the enzyme PFK to
regulate glycolysis. ScPFK also contains homologous α and β subunits and can form stable heterooctamers
α4β4. The internal
sequence duplication α and β subunits similar to that seen in mammalian PFKs
propose that gene duplications lead to a functional diversification of the
catalytic and effector sites, which has allowed eukaryotes to develop more
complex control systems for glycolytic processes. The 6-phosphofructokinase
subunit alpha and 6-phosphofructokinase subunit beta specifically catalyze the
phosphorylation of D-fructose 6-phosphate to fructose 1,6-biphosphae by ATP,
the first committing step of glycolysis. Each α subunit and β subunit can be
divided into two domains, the N-terminal halves and the C-terminal halves, each
resembling the prokaryotic PFK subunit. The interactions between these two
subunits determine the location of the active and effector sites in the
eukaryotic enzyme. When the N-terminal domain of the α subunit interacts with
the N-terminal domain of the β subunit, or when their C-terminal domains behave
in a similar fashion, dimerization occurs.
The alignment of the protein subunits, the binding and
interactions between them the substrate Fru6-P and the activator Fru2,6-P2,
and a comparison with EM studies reveal ScPFK structure to be in the active
state. The N-terminal domain of the eukaryotic PFK subunit retains the
catalytic role, while the C-terminal half has acquired a regulatory function. The relative orientation between the two halves of ScPFK is 75°, whereas the usual
46° observed for the enzyme occurs in the presence of inhibitory amounts of ATP
(5). A β-D-Fru6-P is bounded between the subdomains of the N-terminal half of one
subunit, and the ligand’s 6-phosphate group interacts with two basic side
chains of the neighboring subunit. ATP binding cavities are also found in the
N-terminal domains. The Fru2,6-P2 effector has eight possible sites
on the inner surface of the ScPFK octamer. Each molecule binds in a cavity
between the subdomains of the C-terminal domain of one subunit, while the
ligand’s 6-phosphate group extends across the interface and interacts with two
basic amino acid side chains of the neighboring C-terminal domain. Additionally,
ionic interactions and hydrogen bonds, mainly between the basic amino acid side
chains, take place between the F6P C 988 and the F6P D 982 residues, as well as
between the FDP C 3 and FDP C 4 residues (5). Additionally, there are several
residues that are required for the optimal activity of the activator and
effector on the protein. The Arg-952 and Arg-665 residues in the α subunit and
the Arg-935 and Arg-658 residues in the β subunit form a motif for binding the
activator’s 2-phosphate, and these Arg residues specifically coordinate the phosphate
group. The Glu-694 residue in α and the Glu-688 residue in β is between the Arg
side chains and stabilizes them through hydrogen bonds. Additionally, His-488,
His-481, His-859, and His-853 residues each interact with the 6-phosphate of
the sugar. When protonated, these residues are likely to interfere with the
binding of the sugar ligands and to counteract the stabilizing and activating
effects of the ligands on the enzyme. Also, the mutation of the Asp-543 residue to Ala suggests that the binding of ADP at this site has a functional
role. With the Phosphofructokinase proteins found in Saccharomyces cerevisiae, none were shown to be in alternate
conformations, incorporated in drug complexes, or associate with metal ions.
The secondary structure of ScPFK is composed of 15% beta
sheets, which is equivalent to 30 strands of 122 residues, and 47% alpha
helices, which is equivalent 37 helices of 367 residues (5). PSI Blast is a sequence similarity search
method, in which a query protein or nucleotide sequence is compared to
nucleotide or protein sequences in a target database to identify regions of
local alignment and report those alignments that score, an E value, above a
given score threshold. A score below 0.5 indicates high similarity between
proteins (9). Also, the DALI server was used as a network service for
comparing the protein structure of ScPFK in 3-dimensional. The coordinate of
the query protein structure were submitted and Dali compared them against those
in the Protein Data Bank. It compares tertiary structures of proteins and
calculates the differences in intramolecular distances. A corresponding Z core
was given for each protein; a score above 2 meant that the comparison protein
had similar folds to the ScPFK. Dali server only works for proteins because it
requires the amino acid backbone atoms to make comparison. The structure used
to compare the phosphofructokinase found in Saccharomyces
cerevisiae was the phosphofructokinase with an inhibited T-state (PDB: 6PFK)
found in Geobacillus stearothermophilus. With an E-value of 2e-121 and a Z score of
40.6, this protein was a sufficient choice as the comparison structure because its E value is well below 0.5 and its Z score is well above 2 (2). The
primary sequence of ScPFK, structure, is twice as long (766 residues) compared
to the primary sequence of 6PFK (319 residues) (2). Additionally, they have
nearly identical secondary structure compositions, as 3O8O is 47% helical and
15% beta sheets. When the two different phosphofructokinase structures are superimposed, the structural similarities of the alpha and beta subunits of both molecules can be observed. Furthermore, F6P and FDP are the ligands/prosthetic groups in
3O8O while 2-phosphoglycolic acid is the ligand chemical component in 6PFK. The
crystal structures of 6PFK reveal close coupling between the change of
quaternary structure and local changes triggered by binding of the allosteric
effects. In the tetramer of 6PFK, these concerted changes link all the effector
and substrate sites and accomodate for the change of affinity for the
cooperative substrate.