Pyruvate kinase is the
enzyme involved in the last step of
glycolysis
Glycolysis is the metabolic pathway that converts glucose () into pyruvate (). The free energy released in this process is used to form the high-energy molecules adenosine triphosphate (ATP) and reduced nicotinamide adenine dinucleotide (NADH) ...
. It
catalyzes
Catalysis () is the process of increasing the rate of a chemical reaction by adding a substance known as a catalyst (). Catalysts are not consumed in the reaction and remain unchanged after it. If the reaction is rapid and the catalyst recyc ...
the transfer of a
phosphate group
In chemistry, a phosphate is an anion, salt, functional group or ester derived from a phosphoric acid. It most commonly means orthophosphate, a derivative of orthophosphoric acid .
The phosphate or orthophosphate ion is derived from phospho ...
from
phosphoenolpyruvate
Phosphoenolpyruvate (2-phosphoenolpyruvate, PEP) is the ester derived from the enol of pyruvate and phosphate. It exists as an anion. PEP is an important intermediate in biochemistry. It has the highest-energy phosphate bond found (−61.9 kJ/m ...
(PEP) to
adenosine diphosphate
Adenosine diphosphate (ADP), also known as adenosine pyrophosphate (APP), is an important organic compound in metabolism and is essential to the flow of energy in living cells. ADP consists of three important structural components: a sugar backbon ...
(ADP), yielding one molecule of
pyruvate and one molecule of
ATP. Pyruvate kinase was inappropriately named (inconsistently with a conventional
kinase) before it was recognized that it did not directly catalyze phosphorylation of
pyruvate, which does not occur under physiological conditions. Pyruvate kinase is present in four distinct, tissue-specific isozymes in animals, each consisting of particular kinetic properties necessary to accommodate the variations in metabolic requirements of diverse tissues.
Isozymes in vertebrates
Four
isozymes of pyruvate kinase expressed in vertebrates: L (liver), R (erythrocytes), M1 (muscle and brain) and M2 (early fetal tissue and most adult tissues). The L and R isozymes are expressed by the gene
PKLR, whereas the M1 and M2 isozymes are expressed by the gene
PKM2
Pyruvate kinase isozymes M1/M2 (PKM1/M2), also known as pyruvate kinase muscle isozyme (PKM), pyruvate kinase type K, cytosolic thyroid hormone-binding protein (CTHBP), thyroid hormone-binding protein 1 (THBP1), or opa-interacting protein 3 (OIP3) ...
. The R and L isozymes differ from M1 and M2 in that they are allosterically regulated. Kinetically, the R and L isozymes of pyruvate kinase have two distinct conformation states; one with a high substrate affinity and one with a low substrate affinity. The R-state, characterized by high substrate affinity, serves as the activated form of pyruvate kinase and is stabilized by PEP and
fructose 1,6-bisphosphate (FBP), promoting the glycolytic pathway. The T-state, characterized by low substrate affinity, serves as the inactivated form of pyruvate kinase, bound and stabilized by ATP and
alanine
Alanine (symbol Ala or A), or α-alanine, is an α-amino acid that is used in the biosynthesis of proteins. It contains an amine group and a carboxylic acid group, both attached to the central carbon atom which also carries a methyl group side ...
, causing phosphorylation of pyruvate kinase and the inhibition of glycolysis.
The M2 isozyme of pyruvate kinase can form tetramers or dimers. Tetramers have a high affinity for PEP, whereas, dimers have a low affinity for PEP. Enzymatic activity can be regulated by phosphorylating highly active tetramers of PKM2 into an inactive dimers.
The PKM gene consists of 12
exons and 11
introns. PKM1 and PKM2 are different
splicing products of the M-gene (PKM1 contains exon 9 while PKM2 contains exon 10) and solely differ in 23 amino acids within a 56-amino acid stretch (aa 378-434) at their
carboxy terminus
The C-terminus (also known as the carboxyl-terminus, carboxy-terminus, C-terminal tail, C-terminal end, or COOH-terminus) is the end of an amino acid chain ( protein or polypeptide), terminated by a free carboxyl group (-COOH). When the protein is ...
. The PKM gene is regulated through heterogenous ribonucleotide proteins like hnRNPA1 and hnRNPA2. Human PKM2 monomer has 531 amino acids and is a single chain divided into A, B and C domains. The difference in amino acid sequence between PKM1 and PKM2 allows PKM2 to be allosterically regulated by FBP and for it to form dimers and tetramers while PKM1 can only form tetramers.
Isozymes in bacteria
Many Enterobacteriaceae, including ''
E. coli'', have two isoforms of pyruvate kinase, PykA and PykF, which are 37% identical in ''E. coli'' (Uniprot
PykAPykF. They catalyze the same reaction as in eukaryotes, namely the generation of ATP from ADP and PEP, the last step in
glycolysis
Glycolysis is the metabolic pathway that converts glucose () into pyruvate (). The free energy released in this process is used to form the high-energy molecules adenosine triphosphate (ATP) and reduced nicotinamide adenine dinucleotide (NADH) ...
, a step that is irreversible under physiological conditions. PykF is allosterically regulated by FBP which reflects the central position of PykF in cellular metabolism.
PykF transcription in ''E. coli'' is regulated by the global transcriptional regulator, Cra (FruR). PfkB was shown to be inhibited by MgATP at low concentrations of Fru-6P, and this regulation is important for
gluconeogenesis.
Reaction
Glycolysis
There are two steps in the pyruvate kinase reaction in glycolysis. First, PEP transfers a phosphate group to ADP, producing ATP and the
enolate
In organic chemistry, enolates are organic anions derived from the deprotonation of carbonyl () compounds. Rarely isolated, they are widely used as reagents in the synthesis of organic compounds.
Bonding and structure
Enolate anions are electr ...
of pyruvate. Secondly, a proton must be added to the enolate of pyruvate to produce the functional form of pyruvate that the cell requires. Because the substrate for pyruvate kinase is a simple phospho-sugar, and the product is an ATP, pyruvate kinase is a possible foundation enzyme for the evolution of the glycolysis cycle, and may be one of the most ancient enzymes in all earth-based life. Phosphoenolpyruvate may have been present abiotically, and has been shown to be produced in high yield in a primitive triose glycolysis pathway.
In yeast cells, the interaction of yeast pyruvate kinase (YPK) with PEP and its allosteric effector
Fructose 1,6-bisphosphate (FBP,) was found to be enhanced by the presence of Mg
2+. Therefore, Mg
2+ was concluded to be an important cofactor in the catalysis of PEP into pyruvate by pyruvate kinase. Furthermore, the metal ion Mn
2+ was shown to have a similar, but stronger effect on YPK than Mg
2+. The binding of metal ions to the metal binding sites on pyruvate kinase enhances the rate of this reaction.
The reaction catalyzed by pyruvate kinase is the final step of glycolysis. It is one of three rate-limiting steps of this pathway.
Rate-limiting steps are the slower, regulated steps of a pathway and thus determine the overall rate of the pathway. In glycolysis, the rate-limiting steps are coupled to either the hydrolysis of ATP or the phosphorylation of ADP, causing the pathway to be energetically favorable and essentially irreversible in cells. This final step is highly regulated and deliberately irreversible because pyruvate is a crucial intermediate building block for further metabolic pathways.
Once pyruvate is produced, it either enters the
TCA cycle for further production of ATP under aerobic conditions, or is converted to
lactic acid or
ethanol under anaerobic conditions.
Gluconeogenesis: the reverse reaction
Pyruvate kinase also serves as a regulatory enzyme for
gluconeogenesis, a biochemical pathway in which the liver generates
glucose from pyruvate and other substrates. Gluconeogenesis utilizes noncarbohydrate sources to provide glucose to the brain and red blood cells in times of starvation when direct glucose reserves are exhausted.
During
fasting state
Fasting is the abstention from eating and sometimes drinking. From a purely physiological context, "fasting" may refer to the metabolic status of a person who has not eaten overnight (see "Breakfast"), or to the metabolic state achieved after com ...
, pyruvate kinase is inhibited, thus preventing the "leak-down" of
phosphoenolpyruvate
Phosphoenolpyruvate (2-phosphoenolpyruvate, PEP) is the ester derived from the enol of pyruvate and phosphate. It exists as an anion. PEP is an important intermediate in biochemistry. It has the highest-energy phosphate bond found (−61.9 kJ/m ...
from being converted into pyruvate;
instead, phosphoenolpyruvate is converted into glucose via a cascade of
gluconeogenesis reactions. Although it utilizes similar enzymes, gluconeogenesis is not the reverse of glycolysis. It is instead a pathway that circumvents the irreversible steps of glycolysis. Furthermore, gluconeogenesis and glycolysis do not occur concurrently in the cell at any given moment as they are reciprocally regulated by cell signaling.
Once the gluconeogenesis pathway is complete, the glucose produced is expelled from the liver, providing energy for the vital tissues in the fasting state.
Regulation
Glycolysis is highly regulated at three of its catalytic steps: the phosphorylation of glucose by
hexokinase, the phosphorylation of
fructose-6-phosphate by
phosphofructokinase, and the transfer of phosphate from PEP to ADP by pyruvate kinase. Under wild-type conditions, all three of these reactions are irreversible, have a large negative free energy and are responsible for the regulation of this pathway.
Pyruvate kinase activity is most broadly regulated by allosteric effectors, covalent modifiers and hormonal control. However, the most significant pyruvate kinase regulator is
fructose-1,6-bisphosphate
Fructose 1,6-bisphosphate, also known as Harden-Young ester, is fructose sugar phosphorylated on carbons 1 and 6 (i.e., is a fructosephosphate). The β-D-form of this compound is common in cells. Upon entering the cell, most glucose and fructose ...
(FBP), which serves as an allosteric effector for the enzyme.
Allosteric effectors
Allosteric regulation
In biochemistry, allosteric regulation (or allosteric control) is the regulation of an enzyme by binding an effector molecule at a site other than the enzyme's active site.
The site to which the effector binds is termed the ''allosteric site ...
is the binding of an effector to a site on the protein other than the active site, causing a conformational change and altering the activity of that given protein or enzyme. Pyruvate kinase has been found to be allosterically activated by FBP and allosterically inactivated by ATP and alanine. Pyruvate Kinase tetramerization is promoted by FBP and Serine while tetramer dissociation is promoted by L-Cysteine.
Fructose-1,6-bisphosphate
FBP is the most significant source of regulation because it comes from within the glycolysis pathway. FBP is a glycolytic intermediate produced from the phosphorylation of
fructose 6-phosphate
Fructose 6-phosphate (sometimes called the Neuberg ester) is a derivative of fructose, which has been phosphorylated at the 6-hydroxy group. It is one of several possible fructosephosphates. The β-D-form of this compound is very common in cells. ...
. FBP binds to the allosteric binding site on domain C of pyruvate kinase and changes the conformation of the enzyme, causing the activation of pyruvate kinase activity. As an intermediate present within the glycolytic pathway, FBP provides
feedforward stimulation because the higher the concentration of FBP, the greater the allosteric activation and magnitude of pyruvate kinase activity. Pyruvate kinase is most sensitive to the effects of FBP. As a result, the remainder of the regulatory mechanisms serve as secondary modification.
Covalent modifiers
Covalent modifiers serve as indirect regulators by controlling the phosphorylation, dephosphorylation, acetylation, succinylation and oxidation of enzymes, resulting in the activation and inhibition of enzymatic activity. In the liver,
glucagon and
epinephrine
Adrenaline, also known as epinephrine, is a hormone and medication which is involved in regulating visceral functions (e.g., respiration). It appears as a white microcrystalline granule. Adrenaline is normally produced by the adrenal glands and ...
activate
protein kinase A
In cell biology, protein kinase A (PKA) is a family of enzymes whose activity is dependent on cellular levels of cyclic AMP (cAMP). PKA is also known as cAMP-dependent protein kinase (). PKA has several functions in the cell, including regulatio ...
, which serves as a covalent modifier by phosphorylating and deactivating pyruvate kinase. In contrast, the secretion of insulin in response to blood sugar elevation activates phosphoprotein phosphatase I, causing the dephosphorylation and activation of pyruvate kinase to increase glycolysis. The same covalent modification has the opposite effect on gluconeogenesis enzymes. This regulation system is responsible for the avoidance of a futile cycle through the prevention of simultaneous activation of pyruvate kinase and enzymes that catalyze gluconeogenesis.
Hormonal control
In order to prevent a
futile cycle
A futile cycle, also known as a substrate cycle, occurs when two metabolic pathways run simultaneously in opposite directions and have no overall effect other than to dissipate energy in the form of heat. The reason this cycle was called "futile" c ...
, glycolysis and gluconeogenesis are heavily regulated in order to ensure that they are never operating in the cell at the same time. As a result, the inhibition of pyruvate kinase by glucagon, cyclic AMP and epinephrine, not only shuts down glycolysis, but also stimulates gluconeogenesis. Alternatively, insulin interferes with the effect of glucagon, cyclic AMP and epinephrine, causing pyruvate kinase to function normally and gluconeogenesis to be shut down. Furthermore, glucose was found to inhibit and disrupt gluconeogenesis, leaving pyruvate kinase activity and glycolysis unaffected. Overall, the interaction between hormones plays a key role in the functioning and regulation of glycolysis and gluconeogenesis in the cell.
Inhibitory effect of metformin
Metformin, or
dimethylbiguanide, is the primary treatment used for type 2 diabetes. Metformin has been shown to indirectly affect pyruvate kinase through the inhibition of gluconeogenesis. Specifically, the addition of metformin is linked to a marked decrease in glucose flux and increase in lactate/pyruvate flux from various metabolic pathways. Although metformin does not directly affect pyruvate kinase activity, it causes a decrease in the concentration of ATP. Due to the allosteric inhibitory effects of ATP on pyruvate kinase, a decrease in ATP results in diminished inhibition and the subsequent stimulation of pyruvate kinase. Consequently, the increase in pyruvate kinase activity directs metabolic flux through glycolysis rather than gluconeogenesis.
Gene Regulation
Heterogenous ribonucleotide proteins (hnRNPs) can act on the PKM gene to regulate expression of M1 and M2 isoforms. PKM1 and PKM2 isoforms are splice variants of the PKM gene that differ by a single exon. Various types of hnRNPs such as hnRNPA1 and hnRNPA2 enter the nucleus during hypoxia conditions and modulate expression such that PKM2 is up-regulated. Hormones such as
insulin up-regulate expression of PKM2 while hormones like
tri-iodothyronine (T3) and
glucagon aid in down-regulating PKM2.
Carbohydrate response element binding protein (ChREBP)
ChREBP
Carbohydrate-responsive element-binding protein (ChREBP) also known as MLX-interacting protein-like (MLXIPL) is a protein that in humans is encoded by the ''MLXIPL'' gene. The protein name derives from the protein's interaction with carbohydrate ...
is a
transcription factor that regulates expression of the L isozyme of pyruvate kinase.
A glucose-sensing module contains domains that are targets for regulatory phosphorylation based on the concentrations of glucose and cAMP, which then control its import into the nucleus.
It may also be further activated by directly binding
glucose-6-phosphate. Once in the nucleus, its DNA binding domains activate pyruvate kinase transcription.
Therefore, high glucose and low cAMP causes dephosphorylation of
ChREBP
Carbohydrate-responsive element-binding protein (ChREBP) also known as MLX-interacting protein-like (MLXIPL) is a protein that in humans is encoded by the ''MLXIPL'' gene. The protein name derives from the protein's interaction with carbohydrate ...
, which then upregulates expression of pyruvate kinase in the liver.
Clinical applications
Deficiency
Genetic defects of this enzyme cause the disease known as
pyruvate kinase deficiency. In this condition, a lack of pyruvate kinase slows down the process of glycolysis. This effect is especially devastating in cells that lack
mitochondria, because these cells must use
anaerobic glycolysis
Anaerobic glycolysis is the transformation of glucose to lactate when limited amounts of oxygen (O2) are available. Anaerobic glycolysis is only an effective means of energy production during short, intense exercise, providing energy for a period ...
as their sole source of energy because the
TCA cycle is not available. For example,
red blood cells
Red blood cells (RBCs), also referred to as red cells, red blood corpuscles (in humans or other animals not having nucleus in red blood cells), haematids, erythroid cells or erythrocytes (from Greek ''erythros'' for "red" and ''kytos'' for "holl ...
, which in a state of pyruvate kinase deficiency, rapidly become deficient in ATP and can undergo
hemolysis. Therefore, pyruvate kinase deficiency can cause chronic nonspherocytic
hemolytic anemia (CNSHA).
PK-LR gene mutation
Pyruvate kinase deficiency is caused by an autosomal recessive trait. Mammals have two pyruvate kinase genes, PK-LR (which encodes for pyruvate kinase isozymes L and R) and PK-M (which encodes for pyruvate kinase isozyme M1), but only PKLR encodes for the red blood isozyme which effects pyruvate kinase deficiency. Over 250 PK-LR gene mutations have been identified and associated with pyruvate kinase deficiency. DNA testing has guided the discovery of the location of PKLR on chromosome 1 and the development of direct gene sequencing tests to molecularly diagnose pyruvate kinase deficiency.
Applications of pyruvate kinase inhibition
Reactive Oxygen Species (ROS) Inhibition
Reactive oxygen species (ROS) are chemically reactive forms of oxygen. In human lung cells, ROS has been shown to inhibit the M2 isozyme of pyruvate kinase (PKM2). ROS achieves this inhibition by oxidizing Cys358 and inactivating PKM2. As a result of PKM2 inactivation, glucose flux is no longer converted into pyruvate, but is instead utilized in the pentose phosphate pathway, resulting in the reduction and detoxification of ROS. In this manner, the harmful effects of ROS are increased and cause greater oxidative stress on the lung cells, leading to potential tumor formation. This inhibitory mechanism is important because it may suggest that the regulatory mechanisms in PKM2 are responsible for aiding cancer cell resistance to oxidative stress and enhanced tumorigenesis.
Phenylalanine inhibition
Phenylalanine is found to function as a competitive inhibitor of pyruvate kinase in the brain. Although the degree of phenylalanine inhibitory activity is similar in both fetal and adult cells, the enzymes in the fetal brain cells are significantly more vulnerable to inhibition than those in adult brain cells. A study of PKM2 in babies with the genetic brain disease
phenylketonurics (PKU), showed elevated levels of phenylalanine and decreased effectiveness of PKM2. This inhibitory mechanism provides insight into the role of pyruvate kinase in brain cell damage.
Pyruvate Kinase in Cancer
Cancer cells have characteristically accelerated metabolic machinery and Pyruvate Kinase is believed to have a role in cancer. When compared to healthy cells, cancer cells have elevated levels of the PKM2 isoform, specifically the low activity dimer. Therefore, PKM2 serum levels are used as markers for cancer. The low activity dimer allows for build-up of phosphoenol pyruvate (PEP), leaving large concentrations of glycolytic intermediates for synthesis of biomolecules that will eventually be used by cancer cells.
Phosphorylation of PKM2 by
Mitogen-activated protein kinase 1 (ERK2) causes conformational changes that allow PKM2 to enter the nucleus and regulate glycolytic gene expression required for tumor development. Some studies state that there is a shift in expression from PKM1 to PKM2 during carcinogenesis. Tumor microenvironments like hypoxia activate transcription factors like the hypoxia-inducible factor to promote the transcription of PKM2, which forms a positive feedback loop to enhance its own transcription.
Alternatives
A reversible enzyme with a similar function,
pyruvate phosphate dikinase
Pyruvate, phosphate dikinase, or PPDK () is an enzyme in the family of transferases that catalyzes the chemical reaction
:ATP + pyruvate + phosphate \rightleftharpoons AMP + phosphoenolpyruvate + diphosphate
This enzyme has been studied primar ...
(PPDK), is found in some
bacteria and has been transferred to a number of anaerobic
eukaryote groups (for example, ''
Streblomastix'', ''
Giardia'', ''
Entamoeba
''Entamoeba'' is a genus of Amoebozoa found as internal parasites or commensals of animals.
In 1875, Fedor Lösch described the first proven case of amoebic dysentery in St. Petersburg, Russia. He referred to the amoeba he observed microsco ...
'', and ''
Trichomonas''), it seems via
horizontal gene transfer on two or more occasions. In some cases, the same organism will have both pyruvate kinase and PPDK.
References
External links
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EC 2.7.1
Glycolysis enzymes
Glycolysis