Transketolase
Encyclopedia
Transketolase, an enzyme
of both the pentose phosphate pathway
in animals and the Calvin cycle
of photosynthesis
, catalyzes two important reactions, which operate in opposite directions in these two pathways. In the first reaction of the pentose phosphate pathway, the cofactor thiamine diphosphate accepts a 2-carbon fragment from a 5-carbon ketose (D-xylulose-5-P), then transfers this fragment to a 5-carbon aldose (D-ribose-5-P) to form a 7-carbon ketose (sedoheptulose-7-P). The abstraction of two carbons from D-xylulose-5-P yields the 3-carbon aldose glyceraldehyde-3-P. In the Calvin cycle, transketolase catalyzes the reverse reaction, the conversion of sedoheptulose-7-P and glyceraldehyde-3-P to pentoses, the aldose D-ribose-5-P and the ketose D-xylulose-5-P.
The second reaction catalyzed by transketolase in the pentose phosphate pathway involves the same thiamine diphosphate-mediated transfer of a 2-carbon fragment from D-xylulose-5-P to the aldose erythrose-4-phosphate, affording fructose 6-phosphate
and glyceraldehyde-3-P. Again, in the Calvin cycle exactly the same reaction occurs, but in the opposite direction. Moreover, in the Calvin cycle this is the first reaction catalyzed by transketolase, rather than the second.
In mammals, transketolase connects the pentose phosphate pathway to glycolysis
, feeding excess sugar phosphates into the main carbohydrate metabolic pathways. Its presence is necessary for the production of NADPH, especially in tissues actively engaged in biosyntheses, such as fatty acid synthesis by the liver
and mammary glands, and for steroid
synthesis by the liver
and adrenal glands. Thiamine diphosphate] is an essential cofactor, along with calcium
.
Transketolase is abundantly expressed in the mammalian cornea
by the stromal keratocytes
and epithelial cells and is putatively considered one of the corneal crystallin
s.
for this enzyme is mainly made up of several arginine
, histidine
, serine
, and aspartate side chains, with a glutamate side chain playing a secondary role. These side chains, specifically Arg359, Arg528, His469, and Ser386, are conserved within each transketolase enzyme and interact with the phosphate
group of the donor and acceptor substrates. Because the substrate channel is so narrow, the donor and acceptor substrates cannot be bound simultaneously. Also, the substrates conform into a slightly extended form upon binding in the active site to accommodate this narrow channel.
Although this enzyme is able to bind numerous types of substrates, such as phosphorylated and nonphosphorylated monosaccharides including the keto
and aldosugars fructose
, ribose
, etc., it has a high specificity for the stereoconfiguration of the hydroxyl
groups of the sugars. These hydroxyl groups at C-3 and C-4 of the ketose
donor must be in the D-threo configuration in order to correctly correspond to the C-1 and C-2 positions on the aldose
acceptor. Also they stabilize the substrate in the active site by interacting with the Asp477, His30, and His263 residues. Disruption of this configuration, both the placement of hydroxyl groups or their stereochemistry, would consequently alter the H-bonding between the residues and substrates thus causing a lower affinity for the substrates.
In the first half of this pathway, His263 is used to effectively abstract the C3 hydroxyl proton
which thus allows a 2-carbon segment to be cleaved from fructose 6-phosphate
. The cofactor
necessary for this step to occur is thiamin pyrophosphate (TPP). The binding of TPP to the enzyme incurs no major conformational change to the enzyme; instead, the enzyme has two flexible loops at the active site which make TPP accessible and binding possible. This thus allows the active site to have a "closed" conformation rather than a large conformational change. Later in the pathway, His263 is used as a proton donor for the substrate acceptor-TPP complex which can then generate erythrose-4-phosphate.
The histidine and aspartate side chains are used to effectively stabilize the substrate within the active site and also participate in deprotonation
of the substrate. Specifically, the His 263 and His30 side chains form hydrogen bonds to the aldehyde
end of the substrate, which is deepest into the substrate channel, and Asp477 forms hydrogen bonds with the alpha hydroxyl group on the substrate, where it works to effectively bind the substrate and check for proper stereochemistry. It is also thought that Asp477 could have important catalytic effects because of its orientation in the middle of the active site and its interactions with the alpha hydroxyl group of the substrate. Glu418, which is located in the deepest region of the active site, plays a critical role in stabilizing the TPP cofactor. Specifically, it is involved in the cofactor-assisted proton abstraction from the substrate molecule.
The phosphate group of the substrate also plays an important role in stabilizing the substrate upon its entrance into the active site. The tight ionic
and polar
interactions between this phosphate group and the residues Arg359, Arg528, His469, and Ser386 collectively work to stabilize the substrate by forming H-bonds to the oxygen atoms of the phosphate. The ionic nature is found in the salt bridge
formed from Arg359 to the phosphate group.
then binds to the carbonyl
of the donor substrate thus cleaving the bond between C-2 and C-3. This keto fragment remains covalently bound to the C-2 carbon of TPP. The donor substrate is then released, and the acceptor substrate enters the active site where the fragment, which is bound to the intermediate α-β-dihydroxyethyl thiamin diphosphate, is then transferred to the acceptor.
Experiments have also been conducted which test the effect replacing alanine for the amino acids at the entrance to the active site, Arg359, Arg528, and His469, which interact with the phosphate group of the substrate. This replacement creates a mutant
enzyme with impaired catalytic activity.
. Two diseases are associated with thiamine deficiency: beriberi
and Wernicke-Korsakoff syndrome
. While no mutations could be demonstrated, there is an indication that thiamine deficiency only leads to Wernicke-Korsakoff syndrome in those whose transketolase has a reduced affinity for thiamine. In this way, the activity of transketolase is greatly hindered, and consequently, the entire pentose phosphate pathway is inhibited.
(vitamin B1), and may be used in the diagnosis of Wernicke's encephalopathy
and other B1-deficiency syndromes if the diagnosis is in doubt. Apart from the baseline enzyme activity (which may be normal even in deficiency states), acceleration of enzyme activity after the addition of thiamine pyrophosphate may be diagnostic of thiamine deficiency (0-15% normal, 15-25% deficiency, >25% severe deficiency).
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
of both the pentose phosphate pathway
Pentose phosphate pathway
The pentose phosphate pathway is a process that generates NADPH and pentoses . There are two distinct phases in the pathway. The first is the oxidative phase, in which NADPH is generated, and the second is the non-oxidative synthesis of 5-carbon sugars...
in animals and the Calvin cycle
Calvin cycle
The Calvin cycle or Calvin–Benson-Bassham cycle or reductive pentose phosphate cycle or C3 cycle or CBB cycle is a series of biochemical redox reactions that take place in the stroma of chloroplasts in photosynthetic organisms...
of photosynthesis
Photosynthesis
Photosynthesis is a chemical process that converts carbon dioxide into organic compounds, especially sugars, using the energy from sunlight. Photosynthesis occurs in plants, algae, and many species of bacteria, but not in archaea. Photosynthetic organisms are called photoautotrophs, since they can...
, catalyzes two important reactions, which operate in opposite directions in these two pathways. In the first reaction of the pentose phosphate pathway, the cofactor thiamine diphosphate accepts a 2-carbon fragment from a 5-carbon ketose (D-xylulose-5-P), then transfers this fragment to a 5-carbon aldose (D-ribose-5-P) to form a 7-carbon ketose (sedoheptulose-7-P). The abstraction of two carbons from D-xylulose-5-P yields the 3-carbon aldose glyceraldehyde-3-P. In the Calvin cycle, transketolase catalyzes the reverse reaction, the conversion of sedoheptulose-7-P and glyceraldehyde-3-P to pentoses, the aldose D-ribose-5-P and the ketose D-xylulose-5-P.
The second reaction catalyzed by transketolase in the pentose phosphate pathway involves the same thiamine diphosphate-mediated transfer of a 2-carbon fragment from D-xylulose-5-P to the aldose erythrose-4-phosphate, affording fructose 6-phosphate
Fructose 6-phosphate
Fructose 6-phosphate is fructose sugar phosphorylated on carbon 6 . The β-D-form of this compound is very common in cells. The vast majority of glucose and fructose entering a cell will become converted to this at some point...
and glyceraldehyde-3-P. Again, in the Calvin cycle exactly the same reaction occurs, but in the opposite direction. Moreover, in the Calvin cycle this is the first reaction catalyzed by transketolase, rather than the second.
In mammals, transketolase connects the pentose phosphate pathway to glycolysis
Glycolysis
Glycolysis is the metabolic pathway that converts glucose C6H12O6, into pyruvate, CH3COCOO− + H+...
, feeding excess sugar phosphates into the main carbohydrate metabolic pathways. Its presence is necessary for the production of NADPH, especially in tissues actively engaged in biosyntheses, such as fatty acid synthesis by the liver
Liver
The liver is a vital organ present in vertebrates and some other animals. It has a wide range of functions, including detoxification, protein synthesis, and production of biochemicals necessary for digestion...
and mammary glands, and for steroid
Steroid
A steroid is a type of organic compound that contains a characteristic arrangement of four cycloalkane rings that are joined to each other. Examples of steroids include the dietary fat cholesterol, the sex hormones estradiol and testosterone, and the anti-inflammatory drug dexamethasone.The core...
synthesis by the liver
Liver
The liver is a vital organ present in vertebrates and some other animals. It has a wide range of functions, including detoxification, protein synthesis, and production of biochemicals necessary for digestion...
and adrenal glands. Thiamine diphosphate] is an essential cofactor, along with calcium
Calcium
Calcium is the chemical element with the symbol Ca and atomic number 20. It has an atomic mass of 40.078 amu. Calcium is a soft gray alkaline earth metal, and is the fifth-most-abundant element by mass in the Earth's crust...
.
Transketolase is abundantly expressed in the mammalian cornea
Cornea
The cornea is the transparent front part of the eye that covers the iris, pupil, and anterior chamber. Together with the lens, the cornea refracts light, with the cornea accounting for approximately two-thirds of the eye's total optical power. In humans, the refractive power of the cornea is...
by the stromal keratocytes
Corneal keratocyte
Corneal keratocytes are specialized fibroblasts residing in the stroma. This corneal layer, representing about 85-90% of corneal thickness, is built up from highly regular collagenous lamellae and extracellular matrix components. Keratocytes play the major role in keeping it transparent, healing...
and epithelial cells and is putatively considered one of the corneal crystallin
Crystallin
In anatomy, a crystallin is a water-soluble structural protein found in the lens and the cornea of the eye accounting for the transparency of the structure. It has also been identified in other places such as the heart, and in aggressive breast cancer tumors....
s.
Species distribution
Transketolase is widely expressed in a wide range of organisms including bacteria, plants, and mammals. The following human genes encode proteins with transketolase activity:- TKT (transketolase)
- TKTL1TKTL1Transketolase-like protein 1 is an enzyme that in humans is encoded by the TKTL1 gene.-Further reading:...
(transketolase-like protein 1) - TKTL2TKTL2Transketolase-like protein 2 is an enzyme that in humans is encoded by the TKTL2 gene.-Further reading:...
(transketolase-like protein 1)
Structure
The entrance to the active siteActive site
In biology the active site is part of an enzyme where substrates bind and undergo a chemical reaction. The majority of enzymes are proteins but RNA enzymes called ribozymes also exist. The active site of an enzyme is usually found in a cleft or pocket that is lined by amino acid residues that...
for this enzyme is mainly made up of several arginine
Arginine
Arginine is an α-amino acid. The L-form is one of the 20 most common natural amino acids. At the level of molecular genetics, in the structure of the messenger ribonucleic acid mRNA, CGU, CGC, CGA, CGG, AGA, and AGG, are the triplets of nucleotide bases or codons that codify for arginine during...
, histidine
Histidine
Histidine Histidine, an essential amino acid, has a positively charged imidazole functional group. It is one of the 22 proteinogenic amino acids. Its codons are CAU and CAC. Histidine was first isolated by German physician Albrecht Kossel in 1896. Histidine is an essential amino acid in humans...
, serine
Serine
Serine is an amino acid with the formula HO2CCHCH2OH. It is one of the proteinogenic amino acids. By virtue of the hydroxyl group, serine is classified as a polar amino acid.-Occurrence and biosynthesis:...
, and aspartate side chains, with a glutamate side chain playing a secondary role. These side chains, specifically Arg359, Arg528, His469, and Ser386, are conserved within each transketolase enzyme and interact with the phosphate
Phosphate
A phosphate, an inorganic chemical, is a salt of phosphoric acid. In organic chemistry, a phosphate, or organophosphate, is an ester of phosphoric acid. Organic phosphates are important in biochemistry and biogeochemistry or ecology. Inorganic phosphates are mined to obtain phosphorus for use in...
group of the donor and acceptor substrates. Because the substrate channel is so narrow, the donor and acceptor substrates cannot be bound simultaneously. Also, the substrates conform into a slightly extended form upon binding in the active site to accommodate this narrow channel.
Although this enzyme is able to bind numerous types of substrates, such as phosphorylated and nonphosphorylated monosaccharides including the keto
Keto
Keto can refer to:* The Keto people, an ethnic group of the Siberian North.* Ceto or Keto, a sea goddess in Greek mythology.* Ketone or keto group, the functional group in the chemical compounds ketones....
and aldosugars fructose
Fructose
Fructose, or fruit sugar, is a simple monosaccharide found in many plants. It is one of the three dietary monosaccharides, along with glucose and galactose, that are absorbed directly into the bloodstream during digestion. Fructose was discovered by French chemist Augustin-Pierre Dubrunfaut in 1847...
, ribose
Ribose
Ribose is an organic compound with the formula C5H10O5; specifically, a monosaccharide with linear form H––4–H, which has all the hydroxyl groups on the same side in the Fischer projection....
, etc., it has a high specificity for the stereoconfiguration of the hydroxyl
Hydroxyl
A hydroxyl is a chemical group containing an oxygen atom covalently bonded with a hydrogen atom. In inorganic chemistry, the hydroxyl group is known as the hydroxide ion, and scientists and reference works generally use these different terms though they refer to the same chemical structure in...
groups of the sugars. These hydroxyl groups at C-3 and C-4 of the ketose
Ketose
A ketose is a sugar containing one ketone group per molecule.With 3 carbon atoms, dihydroxyacetone is the simplest of all ketoses and is the only one having no optical activity. Ketoses can isomerize into an aldose when the carbonyl group is located at the end of the molecule...
donor must be in the D-threo configuration in order to correctly correspond to the C-1 and C-2 positions on the aldose
Aldose
An aldose is a monosaccharide that contains only one aldehyde group per molecule. The chemical formula takes the form Cnn. The simplest possible aldose is the diose glycolaldehyde, which only contains two carbon atoms....
acceptor. Also they stabilize the substrate in the active site by interacting with the Asp477, His30, and His263 residues. Disruption of this configuration, both the placement of hydroxyl groups or their stereochemistry, would consequently alter the H-bonding between the residues and substrates thus causing a lower affinity for the substrates.
In the first half of this pathway, His263 is used to effectively abstract the C3 hydroxyl proton
Proton
The proton is a subatomic particle with the symbol or and a positive electric charge of 1 elementary charge. One or more protons are present in the nucleus of each atom, along with neutrons. The number of protons in each atom is its atomic number....
which thus allows a 2-carbon segment to be cleaved from fructose 6-phosphate
Fructose 6-phosphate
Fructose 6-phosphate is fructose sugar phosphorylated on carbon 6 . The β-D-form of this compound is very common in cells. The vast majority of glucose and fructose entering a cell will become converted to this at some point...
. The cofactor
Cofactor (biochemistry)
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the protein's biological activity. These proteins are commonly enzymes, and cofactors can be considered "helper molecules" that assist in biochemical transformations....
necessary for this step to occur is thiamin pyrophosphate (TPP). The binding of TPP to the enzyme incurs no major conformational change to the enzyme; instead, the enzyme has two flexible loops at the active site which make TPP accessible and binding possible. This thus allows the active site to have a "closed" conformation rather than a large conformational change. Later in the pathway, His263 is used as a proton donor for the substrate acceptor-TPP complex which can then generate erythrose-4-phosphate.
The histidine and aspartate side chains are used to effectively stabilize the substrate within the active site and also participate in deprotonation
Deprotonation
Deprotonation is the removal of a proton from a molecule, forming the conjugate base.The relative ability of a molecule to give up a proton is measured by its pKa value. A low pKa value indicates that the compound is acidic and will easily give up its proton to a base...
of the substrate. Specifically, the His 263 and His30 side chains form hydrogen bonds to the aldehyde
Aldehyde
An aldehyde is an organic compound containing a formyl group. This functional group, with the structure R-CHO, consists of a carbonyl center bonded to hydrogen and an R group....
end of the substrate, which is deepest into the substrate channel, and Asp477 forms hydrogen bonds with the alpha hydroxyl group on the substrate, where it works to effectively bind the substrate and check for proper stereochemistry. It is also thought that Asp477 could have important catalytic effects because of its orientation in the middle of the active site and its interactions with the alpha hydroxyl group of the substrate. Glu418, which is located in the deepest region of the active site, plays a critical role in stabilizing the TPP cofactor. Specifically, it is involved in the cofactor-assisted proton abstraction from the substrate molecule.
The phosphate group of the substrate also plays an important role in stabilizing the substrate upon its entrance into the active site. The tight ionic
Ionic bond
An ionic bond is a type of chemical bond formed through an electrostatic attraction between two oppositely charged ions. Ionic bonds are formed between a cation, which is usually a metal, and an anion, which is usually a nonmetal. Pure ionic bonding cannot exist: all ionic compounds have some...
and polar
Chemical polarity
In chemistry, polarity refers to a separation of electric charge leading to a molecule or its chemical groups having an electric dipole or multipole moment. Polar molecules interact through dipole–dipole intermolecular forces and hydrogen bonds. Molecular polarity is dependent on the difference in...
interactions between this phosphate group and the residues Arg359, Arg528, His469, and Ser386 collectively work to stabilize the substrate by forming H-bonds to the oxygen atoms of the phosphate. The ionic nature is found in the salt bridge
Salt bridge
A salt bridge, in chemistry, is a laboratory device used to connect the oxidation and reduction half-cells of a galvanic cell , a type of electrochemical cell...
formed from Arg359 to the phosphate group.
Mechanism
The catalysis of this mechanism is initiated by the deprotonation of TPP at the thiazolium ring. This carbanionCarbanion
A carbanion is an anion in which carbon has an unshared pair of electrons and bears a negative charge usually with three substituents for a total of eight valence electrons. The carbanion exists in a trigonal pyramidal geometry. Formally a carbanion is the conjugate base of a carbon acid.where B...
then binds to the carbonyl
Carbonyl
In organic chemistry, a carbonyl group is a functional group composed of a carbon atom double-bonded to an oxygen atom: C=O. It is common to several classes of organic compounds, as part of many larger functional groups....
of the donor substrate thus cleaving the bond between C-2 and C-3. This keto fragment remains covalently bound to the C-2 carbon of TPP. The donor substrate is then released, and the acceptor substrate enters the active site where the fragment, which is bound to the intermediate α-β-dihydroxyethyl thiamin diphosphate, is then transferred to the acceptor.
Experiments have also been conducted which test the effect replacing alanine for the amino acids at the entrance to the active site, Arg359, Arg528, and His469, which interact with the phosphate group of the substrate. This replacement creates a mutant
Mutant
In biology and especially genetics, a mutant is an individual, organism, or new genetic character, arising or resulting from an instance of mutation, which is a base-pair sequence change within the DNA of a gene or chromosome of an organism resulting in the creation of a new character or trait not...
enzyme with impaired catalytic activity.
Role in disease
Transketolase activity is decreased in deficiency of thiamine, which is generally due to malnutritionMalnutrition
Malnutrition is the condition that results from taking an unbalanced diet in which certain nutrients are lacking, in excess , or in the wrong proportions....
. Two diseases are associated with thiamine deficiency: beriberi
Beriberi
Beriberi is a nervous system ailment caused by a thiamine deficiency in the diet. Thiamine is involved in the breakdown of energy molecules such as glucose and is also found on the membranes of neurons...
and Wernicke-Korsakoff syndrome
Wernicke-Korsakoff syndrome
Wernicke–Korsakoff syndrome is a manifestation of thiamine deficiency, or beriberi. This is usually secondary to alcohol abuse...
. While no mutations could be demonstrated, there is an indication that thiamine deficiency only leads to Wernicke-Korsakoff syndrome in those whose transketolase has a reduced affinity for thiamine. In this way, the activity of transketolase is greatly hindered, and consequently, the entire pentose phosphate pathway is inhibited.
Diagnostic use
Red cell transketolase activity is reduced in deficiency of thiamineThiamine
Thiamine or thiamin or vitamin B1 , named as the "thio-vitamine" is a water-soluble vitamin of the B complex. First named aneurin for the detrimental neurological effects if not present in the diet, it was eventually assigned the generic descriptor name vitamin B1. Its phosphate derivatives are...
(vitamin B1), and may be used in the diagnosis of Wernicke's encephalopathy
Wernicke's encephalopathy
Wernicke encephalopathy is a syndrome characterised by ataxia, ophthalmoplegia, confusion, and impairment of short-term memory.It is caused by lesions in the medial thalamic nuclei, mammillary bodies, periaqueductal and periventricular brainstem nuclei, and superior cerebellar vermis, often...
and other B1-deficiency syndromes if the diagnosis is in doubt. Apart from the baseline enzyme activity (which may be normal even in deficiency states), acceleration of enzyme activity after the addition of thiamine pyrophosphate may be diagnostic of thiamine deficiency (0-15% normal, 15-25% deficiency, >25% severe deficiency).