Glutathione S-transferase
Encyclopedia
Enzymes of the glutathione S-transferase (GST) family are composed of many cytosolic, mitochondrial, and microsomal (now designated as MAPEG
) protein
s. GSTs are present in eukaryotes and in prokaryotes, where they catalyze
a variety of reactions and accept endogenous
and xenobiotic
substrate
s.
GSTs can constitute up to 10% of cytosolic protein in some mammalian organs. GSTs catalyse the conjugation of reduced glutathione
— via a sulfhydryl group — to electrophilic centers on a wide variety of substrates. This activity detoxifies endogenous compounds such as peroxidised lipids, as well as breakdown of xenobiotics. GSTs may also bind toxins and function as transport proteins, and, therefore, an early term for GSTs was “ligandin”.
The mammalian GST super-family consists of cytosolic dimeric isoenzymes of 45–55 kDa
size that have been assigned to at least six classes: Alpha, Mu, Pi, Theta, Zeta and Omega.
Most mammalian isoenzymes have affinity for the substrate 1-chloro-2,4-dinitrobenzene (CDNB), and spectrophotometric assays utilising this substrate are commonly used to report GST activity. However, some endogenous compounds, e.g., bilirubin, can inhibit the activity of GSTs. In mammals, GST isoforms have cell specific distributions (e.g., alpha GST in hepatocytes and pi GST in the biliary tract of the human liver).
ian cytosolic GSTs are dimeric
both subunits being from the same class of GSTs, although not necessarily identical. The monomer
s are in the range of 22–30 kDa. They are active over a wide variety of substrates with considerable overlap.
s. Drugs, poisons, and other compounds not traditionally listed in either groups are usually modified by the phase I and/or phase II mechanisms, and finally excreted from the body. GSTs contribute to this type of metabolism by conjugating these compounds (often electrophilic and somewhat lipophilic
in nature) with reduced glutathione
to facilitate dissolution in the aqueous cellular and extracellular media, and, from there, out of the body.
alongside the gene sequence encoding the protein of interest. Induction of protein expression from the vector's promoter results in expression of a fusion protein: the protein of interest fused to the GST protein. This GST-fusion protein can then be purified from cells via its high affinity for glutathione.
Fusion proteins offer an important biological assay for direct protein-to-protein interactions. For instance, to demonstrate that protein X binds to protein Y a GST-X fusion protein would be generated. Assay
beads, coated with the tripeptide glutathione
, strongly bind the GST fusion protein (GST-X). Therefore if X binds Y, then GST-X will also bind Y, and Y will be present on assay beads.
GST is commonly used to create fusion proteins. The tag has the size of 220 amino acid
s (roughly 26 KDa), which, compared to other tags like the myc
- or the FLAG-tag
, is quite big. It is fused to the N-terminus
of a protein. However, many commercially-available sources of GST-tagged plasmids include a thrombin
domain for cleavage of the GST tag during protein purification.
A GST-tag is often used to separate and purify proteins that contain the GST-fusion. GST-fusion proteins can be produced in Escherichia coli
, as recombinant proteins. The GST part binds its substrate
, glutathione. Agarose beads can be coated with glutathione, and such glutathione-Agarose beads bind GST-proteins. These beads are then washed, to remove contaminating bacterial proteins. Adding free glutathione to beads that bind purified GST-proteins will release the GST-protein in solution.
MAPEG family
MAPEG family of transmembrane proteins with highly divergent functions...
) protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
s. GSTs are present in eukaryotes and in prokaryotes, where they catalyze
Catalysis
Catalysis is the change in rate of a chemical reaction due to the participation of a substance called a catalyst. Unlike other reagents that participate in the chemical reaction, a catalyst is not consumed by the reaction itself. A catalyst may participate in multiple chemical transformations....
a variety of reactions and accept endogenous
Endogenous
Endogenous substances are those that originate from within an organism, tissue, or cell. Endogenous retroviruses are caused by ancient infections of germ cells in humans, mammals and other vertebrates...
and xenobiotic
Xenobiotic
A xenobiotic is a chemical which is found in an organism but which is not normally produced or expected to be present in it. It can also cover substances which are present in much higher concentrations than are usual...
substrate
Substrate (biochemistry)
In biochemistry, a substrate is a molecule upon which an enzyme acts. Enzymes catalyze chemical reactions involving the substrate. In the case of a single substrate, the substrate binds with the enzyme active site, and an enzyme-substrate complex is formed. The substrate is transformed into one or...
s.
GSTs can constitute up to 10% of cytosolic protein in some mammalian organs. GSTs catalyse the conjugation of reduced glutathione
Glutathione
Glutathione is a tripeptide that contains an unusual peptide linkage between the amine group of cysteine and the carboxyl group of the glutamate side-chain...
— via a sulfhydryl group — to electrophilic centers on a wide variety of substrates. This activity detoxifies endogenous compounds such as peroxidised lipids, as well as breakdown of xenobiotics. GSTs may also bind toxins and function as transport proteins, and, therefore, an early term for GSTs was “ligandin”.
The mammalian GST super-family consists of cytosolic dimeric isoenzymes of 45–55 kDa
Atomic mass unit
The unified atomic mass unit or dalton is a unit that is used for indicating mass on an atomic or molecular scale. It is defined as one twelfth of the rest mass of an unbound neutral atom of carbon-12 in its nuclear and electronic ground state, and has a value of...
size that have been assigned to at least six classes: Alpha, Mu, Pi, Theta, Zeta and Omega.
Most mammalian isoenzymes have affinity for the substrate 1-chloro-2,4-dinitrobenzene (CDNB), and spectrophotometric assays utilising this substrate are commonly used to report GST activity. However, some endogenous compounds, e.g., bilirubin, can inhibit the activity of GSTs. In mammals, GST isoforms have cell specific distributions (e.g., alpha GST in hepatocytes and pi GST in the biliary tract of the human liver).
Family members
The following is a list of human glutathione S-transferases:| Class | Members |
|---|---|
| alpha | GSTA1, GSTA2 GSTA2 Glutathione S-transferase A2 is an enzyme that in humans is encoded by the GSTA2 gene.-Further reading:... , GSTA3 GSTA3 Glutathione S-transferase A3 is an enzyme that in humans is encoded by the GSTA3 gene.-Further reading:... , GSTA4 GSTA4 Glutathione S-transferase A4, also known as GSTA4, is an enzyme which in humans is encoded by the GSTA4 gene.- Function :Cytosolic and membrane-bound forms of glutathione S-transferase are encoded by two distinct supergene families. These enzymes are involved in cellular defense against toxic,... , GSTA5 |
| kappa | GSTK1 GSTK1 Glutathione S-transferase kappa 1 is an enzyme that in humans is encoded by the GSTK1 gene.-Further reading:... |
| mu | GSTM1 Glutathione S-transferase Mu 1 Glutathione S-transferase Mu 1 is a human glutathione S-transferase.-Further reading:... , GSTM1L, GSTM2 GSTM2 Glutathione S-transferase Mu 2 is an enzyme that in humans is encoded by the GSTM2 gene.-Further reading:... , GSTM3 GSTM3 Glutathione S-transferase M3 , also known as GSTM3, is an enzyme which in humans is encoded by the GSTM3 gene.- Function :Cytosolic and membrane-bound forms of glutathione S-transferase are encoded by two distinct supergene families... , GSTM4 GSTM4 Glutathione S-transferase Mu 4 is an enzyme that in humans is encoded by the GSTM4 gene.In the August 2009 issue of Oncogene journal, researchers at Huntsman Cancer Institute at the University of Utah demonstrated that expression levels of GSTM4 could predict response to chemotherapy in patients... , GSTM5 |
| omega | GSTO1 GSTO1 Glutathione S-transferase omega-1 is an enzyme that in humans is encoded by the GSTO1 gene.-Further reading:... , GSTO2 GSTO2 Glutathione S-transferase omega-2 is an enzyme that in humans is encoded by the GSTO2 gene.-Further reading:... |
| pi | GSTP1 GSTP1 Glutathione S-transferase P is an enzyme that in humans is encoded by the GSTP1 gene.-Interactions:GSTP1 has been shown to interact with Fanconi anemia, complementation group C and MAPK8.-Further reading:... |
| theta | GSTT1 GSTT1 Glutathione S-transferase theta-1 is an enzyme that in humans is encoded by the GSTT1 gene.-References:... , GSTT2 GSTT2 Glutathione S-transferase theta-2 is an enzyme that in humans is encoded by the GSTT2 gene.-Further reading:... |
| zeta | GSTZ1 GSTZ1 Maleylacetoacetate isomerase is an enzyme that in humans is encoded by the GSTZ1 gene.-Further reading:... |
| microsomal | MGST1, MGST2 MGST2 Microsomal glutathione S-transferase 2 is an enzyme that in humans is encoded by the MGST2 gene.-Further reading:... , MGST3 MGST3 Microsomal glutathione S-transferase 3 is an enzyme that in humans is encoded by the MGST3 gene.-Further reading:... |
Structure
MammalMammal
Mammals are members of a class of air-breathing vertebrate animals characterised by the possession of endothermy, hair, three middle ear bones, and mammary glands functional in mothers with young...
ian cytosolic GSTs are dimeric
Protein dimer
In biochemistry, a dimer is a macromolecular complex formed by two, usually non-covalently bound, macromolecules like proteins or nucleic acids...
both subunits being from the same class of GSTs, although not necessarily identical. The monomer
Monomer
A monomer is an atom or a small molecule that may bind chemically to other monomers to form a polymer; the term "monomeric protein" may also be used to describe one of the proteins making up a multiprotein complex...
s are in the range of 22–30 kDa. They are active over a wide variety of substrates with considerable overlap.
GSTs and biotransformation
Glutathione S-transferases are considered, among several others, to contribute to the phase II biotransformation of xenobioticXenobiotic
A xenobiotic is a chemical which is found in an organism but which is not normally produced or expected to be present in it. It can also cover substances which are present in much higher concentrations than are usual...
s. Drugs, poisons, and other compounds not traditionally listed in either groups are usually modified by the phase I and/or phase II mechanisms, and finally excreted from the body. GSTs contribute to this type of metabolism by conjugating these compounds (often electrophilic and somewhat lipophilic
Lipophilic
Lipophilicity, , refers to the ability of a chemical compound to dissolve in fats, oils, lipids, and non-polar solvents such as hexane or toluene. These non-polar solvents are themselves lipophilic — the axiom that like dissolves like generally holds true...
in nature) with reduced glutathione
Glutathione
Glutathione is a tripeptide that contains an unusual peptide linkage between the amine group of cysteine and the carboxyl group of the glutamate side-chain...
to facilitate dissolution in the aqueous cellular and extracellular media, and, from there, out of the body.
GST-tags and the GST pull-down assay
Genetic engineers have used glutathione S-transferase to create the GST gene fusion system. This system is used to purify and detect proteins of interest. In a GST gene fusion system, the GST sequence is incorporated into an expression vectorExpression vector
An expression vector, otherwise known as an expression construct, is generally a plasmid that is used to introduce a specific gene into a target cell. Once the expression vector is inside the cell, the protein that is encoded by the gene is produced by the cellular-transcription and translation...
alongside the gene sequence encoding the protein of interest. Induction of protein expression from the vector's promoter results in expression of a fusion protein: the protein of interest fused to the GST protein. This GST-fusion protein can then be purified from cells via its high affinity for glutathione.
Fusion proteins offer an important biological assay for direct protein-to-protein interactions. For instance, to demonstrate that protein X binds to protein Y a GST-X fusion protein would be generated. Assay
Assay
An assay is a procedure in molecular biology for testing or measuring the activity of a drug or biochemical in an organism or organic sample. A quantitative assay may also measure the amount of a substance in a sample. Bioassays and immunoassays are among the many varieties of specialized...
beads, coated with the tripeptide glutathione
Glutathione
Glutathione is a tripeptide that contains an unusual peptide linkage between the amine group of cysteine and the carboxyl group of the glutamate side-chain...
, strongly bind the GST fusion protein (GST-X). Therefore if X binds Y, then GST-X will also bind Y, and Y will be present on assay beads.
GST is commonly used to create fusion proteins. The tag has the size of 220 amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
s (roughly 26 KDa), which, compared to other tags like the myc
Myc
Myc is a regulator gene that codes for a transcription factor. In the human genome, Myc is located on chromosome 8 and is believed to regulate expression of 15% of all genes through binding on Enhancer Box sequences and recruiting histone acetyltransferases...
- or the FLAG-tag
FLAG-tag
FLAG-tag, or FLAG octapeptide, is a polypeptide protein tag that can be added to a protein using recombinant DNA technology. It can be used for affinity chromatography, then used to separate recombinant, overexpressed protein from wild-type protein expressed by the host organism...
, is quite big. It is fused to the N-terminus
N-terminal end
The N-terminus refers to the start of a protein or polypeptide terminated by an amino acid with a free amine group . The convention for writing peptide sequences is to put the N-terminus on the left and write the sequence from N- to C-terminus...
of a protein. However, many commercially-available sources of GST-tagged plasmids include a thrombin
Thrombin
Thrombin is a "trypsin-like" serine protease protein that in humans is encoded by the F2 gene. Prothrombin is proteolytically cleaved to form thrombin in the first step of the coagulation cascade, which ultimately results in the stemming of blood loss...
domain for cleavage of the GST tag during protein purification.
A GST-tag is often used to separate and purify proteins that contain the GST-fusion. GST-fusion proteins can be produced in Escherichia coli
Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
, as recombinant proteins. The GST part binds its substrate
Substrate (biochemistry)
In biochemistry, a substrate is a molecule upon which an enzyme acts. Enzymes catalyze chemical reactions involving the substrate. In the case of a single substrate, the substrate binds with the enzyme active site, and an enzyme-substrate complex is formed. The substrate is transformed into one or...
, glutathione. Agarose beads can be coated with glutathione, and such glutathione-Agarose beads bind GST-proteins. These beads are then washed, to remove contaminating bacterial proteins. Adding free glutathione to beads that bind purified GST-proteins will release the GST-protein in solution.
Role in disease
Genetic polymorphisms in glutathione S-transferase and its altered expression and activity are associated with oxidative DNA damage and also damage of the kidney. Subsequently the individual’s risk of cancer susceptibility increases.See also
- Maltose-binding protein
- Glutathione S-transferase, C-terminal domainGlutathione S-transferase, C-terminal domainGlutathione S-transferase, C-terminal domain is a structural domain of glutathione S-transferase .GST conjugates reduced glutathione to a variety of targets includingS-crystallin from squid, the eukaryotic elongation factor...
- Bacterial glutathione transferaseBacterial glutathione transferaseBacterial glutathione transferases are part of a superfamily of enzymes that play a crucial role in cellular detoxification...
- Affinity chromatographyAffinity chromatographyAffinity chromatography is a method of separating biochemical mixtures and based on a highly specific interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand.-Uses:Affinity chromatography can be used to:...
External links
- Overview of Glutathione-S-Transferases - MAPEG (Eicosanoid and Glutathione metabolism proteins) family
- Preparation of GST Fusion Proteins