Transketolase (abbreviated as TK) is an
enzyme
Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different molecule ...
that is encoded by the TKT
gene
In biology, the word gene (from , ; "... Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a b ...
. It participates in both the
pentose phosphate pathway in all organisms and the
Calvin cycle
The Calvin cycle, light-independent reactions, bio synthetic phase, dark reactions, or photosynthetic carbon reduction (PCR) cycle of photosynthesis is a series of chemical reactions that convert carbon dioxide and hydrogen-carrier compounds into ...
of
photosynthesis
Photosynthesis is a process used by plants and other organisms to convert light energy into chemical energy that, through cellular respiration, can later be released to fuel the organism's activities. Some of this chemical energy is stored i ...
. Transketolase catalyzes two important reactions, which operate in opposite directions in these two pathways. In the first reaction of the non-oxidative 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
Nicotinamide adenine dinucleotide phosphate, abbreviated NADP or, in older notation, TPN (triphosphopyridine nucleotide), is a cofactor used in anabolic reactions, such as the Calvin cycle and lipid and nucleic acid syntheses, which require NADP ...
, especially in tissues actively engaged in biosyntheses, such as fatty acid synthesis by the
liver
The liver is a major organ only found in vertebrates which performs many essential biological functions such as detoxification of the organism, and the synthesis of proteins and biochemicals necessary for digestion and growth. In humans, it ...
and
mammary glands
A mammary gland is an exocrine gland in humans and other mammals that produces milk to feed young offspring. Mammals get their name from the Latin word ''mamma'', "breast". The mammary glands are arranged in organs such as the breasts in ...
, and for
steroid
A steroid is a biologically active organic compound with four rings arranged in a specific molecular configuration. Steroids have two principal biological functions: as important components of cell membranes that alter membrane fluidity; and ...
synthesis by the
liver
The liver is a major organ only found in vertebrates which performs many essential biological functions such as detoxification of the organism, and the synthesis of proteins and biochemicals necessary for digestion and growth. In humans, it ...
and
adrenal glands
The adrenal glands (also known as suprarenal glands) are endocrine glands that produce a variety of hormones including adrenaline and the steroids aldosterone and cortisol. They are found above the kidneys. Each gland has an outer cortex wh ...
. Thiamine diphosphate is an essential cofactor, along with
calcium
Calcium is a chemical element with the symbol Ca and atomic number 20. As an alkaline earth metal, calcium is a reactive metal that forms a dark oxide-nitride layer when exposed to air. Its physical and chemical properties are most similar t ...
.
Transketolase is abundantly expressed in the mammalian
cornea
The cornea is the transparent front part of the eye that covers the iris, pupil, and anterior chamber. Along with the anterior chamber and lens, the cornea refracts light, accounting for approximately two-thirds of the eye's total optical ...
by the
stromal keratocytes and epithelial cells and is reputed to be one of the corneal
crystallins.
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)
* ''
TKTL1
Transketolase-like-1 (TKTL1) is a gene closely related to the transketolase gene (TKT). It emerged in mammals during the course of evolution and, according to the latest research findings, is considered one of the key genes that distinguishes mod ...
'' (transketolase-like protein 1)
* ''
TKTL2'' (transketolase-like protein 2)
Structure
The entrance to the
active site for this enzyme is made up mainly of several
arginine
Arginine is the amino acid with the formula (H2N)(HN)CN(H)(CH2)3CH(NH2)CO2H. The molecule features a guanidino group appended to a standard amino acid framework. At physiological pH, the carboxylic acid is deprotonated (−CO2−) and both the a ...
,
histidine
Histidine (symbol His or H) is an essential amino acid that is used in the biosynthesis of proteins. It contains an α-amino group (which is in the protonated –NH3+ form under biological conditions), a carboxylic acid group (which is in the ...
,
serine
Serine (symbol Ser or S) is an α-amino acid that is used in the biosynthesis of proteins. It contains an α- amino group (which is in the protonated − form under biological conditions), a carboxyl group (which is in the deprotonated − for ...
, and
aspartate
Aspartic acid (symbol Asp or D; the ionic form is known as aspartate), is an α-amino acid that is used in the biosynthesis of proteins. Like all other amino acids, it contains an amino group and a carboxylic acid. Its α-amino group is in the pro ...
side-chains, with a
glutamate
Glutamic acid (symbol Glu or E; the ionic form is known as glutamate) is an α-amino acid that is used by almost all living beings in the biosynthesis of proteins. It is a non-essential nutrient for humans, meaning that the human body can syn ...
side-chain playing a secondary role. These side-chains, to be specific Arg359, Arg528, His469, and Ser386, are conserved within each transketolase enzyme and interact with the
phosphate
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 phosph ...
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
monosaccharide
Monosaccharides (from Greek '' monos'': single, '' sacchar'': sugar), also called simple sugars, are the simplest forms of sugar and the most basic units (monomers) from which all carbohydrates are built.
They are usually colorless, water-sol ...
s including the
keto and aldosugars
fructose
Fructose, or fruit sugar, is a ketonic simple sugar found in many plants, where it is often bonded to glucose to form the disaccharide sucrose. It is one of the three dietary monosaccharides, along with glucose and galactose, that are absorb ...
,
ribose
Ribose is a simple sugar and carbohydrate with molecular formula C5H10O5 and the linear-form composition H−(C=O)−(CHOH)4−H. The naturally-occurring form, , is a component of the ribonucleotides from which RNA is built, and so this compou ...
, etc., it has a high specificity for the stereoconfiguration of the
hydroxyl
In chemistry, a hydroxy or hydroxyl group is a functional group with the chemical formula and composed of one oxygen atom covalently bonded to one hydrogen atom. In organic chemistry, alcohols and carboxylic acids contain one or more hydrox ...
groups of the sugars. These hydroxyl groups at C-3 and C-4 of the
ketose
A ketose is a monosaccharide containing one ketone group per molecule. The simplest ketose is dihydroxyacetone, which has only three carbon atoms. It is the only ketose with no optical activity. All monosaccharide ketoses are reducing sugars, be ...
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
Thiamine pyrophosphate (TPP or ThPP), or thiamine diphosphate (ThDP), or cocarboxylase is a thiamine (vitamin B1) derivative which is produced by the enzyme thiamine diphosphokinase. Thiamine pyrophosphate is a cofactor that is present in all liv ...
(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 that make TPP accessible and binding possible.
[ Thus, this 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 (or dehydronation) is the removal (transfer) of a proton (or hydron, or hydrogen cation), (H+) from a Brønsted–Lowry acid in an acid–base reaction.Henry Jakubowski, Biochemistry Online Chapter 2A3, https://employees.csbsju. ...
of the substrate. To be specific, the His 263 and His30 side-chains form hydrogen bonds to the aldehyde
In organic chemistry, an aldehyde () is an organic compound containing a functional group with the structure . The functional group itself (without the "R" side chain) can be referred to as an aldehyde but can also be classified as a formyl group ...
end of the substrate, which is deepest into the substrate channel, and Asp477 forms hydrogen bonds
In chemistry, a hydrogen bond (or H-bond) is a primarily electrostatic force of attraction between a hydrogen (H) atom which is covalently bound to a more electronegative "donor" atom or group (Dn), and another electronegative atom bearing a ...
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. To be specific, 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.
]
Mechanism
The catalysis of this mechanism is initiated by the deprotonation of TPP at the thiazolium ring. This carbanion
In organic chemistry, a carbanion is an anion in which carbon is trivalent (forms three bonds) and bears a formal negative charge (in at least one significant resonance form).
Formally, a carbanion is the conjugate base of a carbon acid:
:R3CH ...
then binds to the 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. A compound containin ...
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 that 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
In biology, and especially in genetics, a mutant is an organism or a new genetic character arising or resulting from an instance of mutation, which is generally an alteration of the DNA sequence of the genome or chromosome of an organism. It ...
enzyme with impaired catalytic activity.[
]
Role in disease
Transketolase activity is decreased in deficiency of thiamine, which in general is due to malnutrition
Malnutrition occurs when an organism gets too few or too many nutrients, resulting in health problems. Specifically, it is "a deficiency, excess, or imbalance of energy, protein and other nutrients" which adversely affects the body's tissues ...
. Several diseases are associated with thiamine deficiency, including beriberi
Thiamine deficiency is a medical condition of low levels of thiamine (Vitamin B1). A severe and chronic form is known as beriberi. The two main types in adults are wet beriberi and dry beriberi. Wet beriberi affects the cardiovascular system, r ...
, biotin-thiamine-responsive basal ganglia disease (BTBGD), Wernicke–Korsakoff syndrome
Wernicke–Korsakoff syndrome (WKS) is the combined presence of Wernicke encephalopathy (WE) and Korsakoff syndrome. Due to the close relationship between these two disorders, people with either are usually diagnosed with WKS as a single syndro ...
, and others (see thiamine
Thiamine, also known as thiamin and vitamin B1, is a vitamin, an essential micronutrient, that cannot be made in the body. It is found in food and commercially synthesized to be a dietary supplement or medication. Phosphorylated forms of t ...
for a comprehensive listing).
In Wernicke–Korsakoff syndrome, while no mutations could be demonstrated, there is an indication that thiamine deficiency leads to Wernicke–Korsakoff syndrome only in those whose transketolase has a reduced affinity for thiamine. In this way, the activity of transketolase is greatly hindered, and, as a consequence, the entire pentose phosphate pathway is inhibited.
Diagnostic use
Red blood cell
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 ...
transketolase activity is reduced in deficiency of thiamine
Thiamine, also known as thiamin and vitamin B1, is a vitamin, an essential micronutrient, that cannot be made in the body. It is found in food and commercially synthesized to be a dietary supplement or medication. Phosphorylated forms of t ...
(vitamin B1), and may be used in the diagnosis of Wernicke 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).
References
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Photosynthesis
EC 2.2.1