Glutathione S-transferase



The glutathione S-transferase (GST) family of enzymes comprises a long list of cytosolic, mitochondrial, and microsomal proteins which are capable of multiple reactions with a multitude of substrates, both endogenous and xenobiotic.

These enzymes can constitute up to 10% of cytosolic protein in some mammalian organs. GSTs catalyse the conjugation of reduced glutathione via the sulfhydryl group, to electrophilic centres on a wide variety of substrates. This activity is useful in the detoxification of endogenous compounds such as peroxidised lipids as well as the metabolism of xenobiotics. As well as their enzymatic activities, GSTs may also bind toxins and function as transport proteins. Because of this, an early term for GSTs was “ligandin”.

The mammalian GST super-family comprises cytosolic dimeric isoenzymes of 45–55 kDa size which have been assigned to at least four generic classes: Alpha, Mu, Pi and Theta. Most mammalian isoenzymes have activity 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 materials, e,g., bilirubin, can inhibit the enzyme activity of GSTs. Immunoassay techniques avoid this problem plus, by the use of class specific antisera, they enable different GST classes to be simultaneously and separately quantified in biological fluids. 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). .

Structure
Mammalian cytosolic GSTs are dimeric both subunits being from the same class of GSTs, although not necessarily identical. The monomers are in the range of 22–29 kDa (see the X-ray structure of the monomer on your right). 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 xenobiotics. Drugs, poisons, and other compounds not traditionally listed in either groups are usually somewhat 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 extracelluar media, and from there, out of the body.

GST-tag
Genetic engineers have used Glutathione S-transferase to create the so-called 'GST gene fusion system'. Here, GST is used to express, purify and detect proteins of interest. In a GST gene fusion system, the GST sequence is incorporated into an expression vector alongside the gene sequence encoding the protein of interest. Induction of protein expression from the vector's multiple cloning sites results in expression of a fusion protein - the protein of interest fused to the GST protein, which can then be released from the cells and purified via its high affinity for glutathione.

GST is commonly used to create fusion proteins. The tag has the size of 220 amino acids, 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.