NEBRASKA REDOX BIOLOGY CENTER EDUCATIONAL PORTAL

Nebraska Redox Biology Center Educational Portal


Glutathione S-transferases

Glutathione S-transferases (GSTs) are superfamily of enzymes that are involved in protein glutathionylation and detoxication of xenobiotics [ 1, 2, 3, 4 ]. They also catalyze specific reactions in a number of biosynthetic and catabolic pathways and play an important role in defense against oxidative stress by reducing peroxides and dehydroascorbates. GSTs are conjugating the thiol group of the glutathione to electrophilic xenobiotics and protecting cells against the mutagenic, carcinogenic, and toxic effects of such compounds. GSTs activity was found in eukaryotes and bacterias [ 1, 2, 5, 6, 7 ]. There are 37 solved Glutathione S-transferase structures in Protein Data Bank at this time (June 2015).


3D structure of Human Glutathione S-transferase. Thioredoxin folsd domain is responsable for glutathione binding and alpha-helical domain is involved in binding of electrophilic sustrate.,


There are three structurally distinct GST superfamilies. Two of them have wide range substrate specificity and act by promoting of GSH for nucleophilic attack on an electrophilic substrate [ 1, 2, 3, 5 ].

1) B1a. Cytoplasmic GSTs. This type of GSTs are found in the cytoplasm of eukaryotes and bacteria. The cytosolic GSTs are dimeric enzymes. Each subunit contains an N-terminal thioredoxin fold. he GSH binding site is located primarily in the thioredoxin fold N-terminal domain and electrophilic substrate binding site is located in the C-terminal domain. GSH binding to GST is resulting on decrease of its pKa from 8.5 to about 6-7 due to selective stabilization of the thiolate form. The cytosolic GST superfamily is divided into several classes. Enzymes within a class generally share more than 40% sequence identity, while enzymes in different classes share less than 25% sequence identity. All of these enzymes share the same fold [ 1, 2, 3, 5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 ].


2) B1b Mitochondrial GSTs. Structurally distinct kappa class of GST is localizesd in mitochondria of eukaryotic cells. Kappa GSTs cantain electrofilic substrate binding alpha-helical domain inserted in the middle of the thioredoxin-fold domain and have strong structural similarity to disulfide isomerase DsbA. Biological function of this class of GSTs is not known at this time [ 1, 2, 19, 20 ].

3) B1c. The MAPEG superfamily. A third type of GST is found in the "membrane-associated proteins in eicosanoid and GSH" metabolism (MAPEG) superfamily. Eicosanoids are c ompounds derived from 20-carbon fatty acids and include prostaglandins, prostacyclins, and leukotrienes. MGST1 is the best characterized enzyme, which exist as monomers or trimer. This superfamily GSTs are found in eukaryotes and some bacteria There are six families of MAPEG proteins, including three families of microsomal GSTs (MGST1, MGST2, and MGST3), leukotri ene C4 synthase, prostaglandin E synthase, and 5-lipoxygenase activating protein (FLAP) [ 1, 2, 21, 22 ].

B1a. Cytoplasmic glutathione S-transferases:

>gi|83406048|gb|AAI10892.1| Glutathione S-transferase ALPHA [Homo sapiens]
MAEKPKLHYFNARGRMESTRWLLAAAGVEFEEKFIKSAEDLDKLRNDGYLMFQQVPMVEIDGMKLVQTRAILNYIASKYNLYGKDIKERALIDMYIEGIADLGEMILL
LPVCPPEEKDAKLALIKEKIKNRYFPAFEKVLKSHGQDYLVGNKLSRADIHLVELLYYVEELDSSLISSFPLLKALKTRISNLPTVKKFLQPGSPRKPPMDEKSLEEA
RKIFRF


>gi|597515728|emb|CDO14821.1| Glutathione S-transferase BETA [Klebsiella pneumoniae]
MKLFYKPGACSLASHIALRESGLDFTLQSVDLAQKRLENGEDYLLINPKGQVPALLLDDDILLTEGVAIMQYIADQVPDRHLLAPVGSIARYQTLEWLNYVATELHKS
FTPLFRPDTPEDYKPVARGLLEKKLQYVDAALADKQWLTGHRFTIADGYLFTVLRWAYAIKLDMAAYGHIGSWMTQVAARPVVAAALAAEGLK


>gi|386306399|gb|AFJ05093.1| Glutathione S-transferase DELTA [Bactrocera dorsalis]
MDFYYMPASAPCRAVLLTAKCVGIELNKILLDLRAGEHLTPEFLKINPQHTIPTLVDKDFALWESRAIMIYLVEQYAKTDSLYSQCPETRALINQRLYFDMNLALTFG
KYFYKPVMSKTPFDPEQRKQLETQLELFNTLLAGNNFVIGETLTLADLALLATISTIDVAQCLKDFNVNVRKYAHIQKWYENMRAVTPGFKENQEGCLEMKKFFEGQ


>gi|386306401|gb|AFJ05094.1| Glutathione S-transferase EPSILON [Bactrocera dorsalis]
MSSKPVLYYTLRSPPCRAVLLTAAAIGLELELRLTNLKERDHLTPEFLKLNPQHTIPVLDDNGTVVTDSHVINAYLVDKYSSDETLYPKDQQKRREVDARLYFDAGHL
FPRVRLMVEPVIYFGADKIQQEKIAYMQLAYDGLEKCLANAPYLCGEHLTIADLCAVASVSSAVHFAPIDEEKFPQLAAWLKRMSLLPYYKKSNQEGADLLGSFVKEQ
MAANKKAKEAEK


>gi|386306411|gb|AFJ05099.1| Glutathione S-transferase THETA [Bactrocera dorsalis]
MQPVKFYFSFLDQSSRALYILLEASKIPFEAIPISILKGEHLTGEFRDKVSQFKCLPVINDDNLSLVGSITIFRHLNREKIVPEHWYPRRNYGRSRIDEFLEWQQRNT
AMACGNYFQQKWLSPILNKASPDAKNASVLTECLNQSLKNFENHFLNKNKFVIGENISYADLMAICEIDQPKFIGFDPFNHHPKLGKWYDRVREELGPYYKKVAIEFD
NKLRTAEKKIPEVMYLQQ


>gi|386306413|gb|AFJ05100.1| Glutathione S-transferase ZETA [Bactrocera dorsalis]
MKPILYSYWHSSCSWRVRTALHWKNIPYETRAVNLLKPESGQHSAEYLAINPTAHVPTLFIDGKNIIESIAILHYLEETRPLPALLPQDAYERAKVREIVEIIASGIQ
PLPNRKVQKRVEHDKRLEWVQHWVNSGFRALEEKLYTTAGKYCVGDEVSMADCCLLPQVFNARNSQVDMRQYPIISRIVSELEMIPAFIAAHPHNQPDCPTELSGK


>gi|23065544|ref|NP_000552.2| Glutathione S-transferase MU [Homo sapiens]
MPMILGYWDIRGLAHAIRLLLEYTDSSYEEKKYTMGDAPDYDRSQWLNEKFKLGLDFPNLPYLIDGAHKITQSNAILCYIARKHNLCGETEEEKIRVDILENQTMDNH
MQLGMICYNPEFEKLKPKYLEELPEKLKLYSEFLGKRPWFAGNKITFVDFLVYDVLDLHRIFEPKCLDAFPNLKDFISRFEGLEKISAYMKSSRFLPRPVFSKMAVWG
NK


>gi|15012036|gb|AAH10915.1| Glutathione S-transferase PI [Homo sapiens]
MPPYTVVYFPVRGRCAALRMLLADQGQSWKEEVVTVETWQEGSLKASCLYGQLPKFQDGDLTLYQSNTILRHLGRTLGLYGKDQQEAALVDMVNDGVEDLRCKYVSLI
YTNYEAGKDDYVKALPGQLKPFETLLSQNQGGKTFIVGDQISFADYNLLDLLLIHEVLAPGCLDAFPLLSAYVGRLSARPKLKAFLASPEYVNLPINGNGKQ


>gi|440214466|gb|AGB93572.1| Glutathione S-transferase SIGMA [Drosophila melanogaster]
MADEAQAPPAEGAPPAEGEAPPPAEGAEGAVEGGEAAPPAEPAEPIKHSYTLFYFNVKALAEPLRYLFAYGNQEYEDVRVTRDEWPALKPTMPMGQMPVLEVDGKRVH
QSISMARFLAKTVGLCGATPWEDLQIDIVVDTINDFRLSSEQFVSYEPEDEIKEKKLVTLNAEVIPFYLEKLEQTVKDNDGHLALGKLTWADVYFAGITDYMNYMVKR
DLLEPYPALRGVVDAVNALEPIKAWIEKRPVTEV


>gi|332191433|gb|AEE29554.1| Glutathione S-transferase TAU [Arabidopsis thaliana]
MADEVILLDFWASMFGMRTRIALAEKRVKYDHREEDLWNKSSLLLEMNPVHKKIPVLIHNGKPVCESLIQIEYIDETWPDNNPLLPSDPYKRAHAKFWADFIDKKVNV
TARRIWAVKGEEQEAAKELIEILKTLESELGDKKYFGDETFGYVDIALIGFHSWFAVYEKFGNVSIESECSKLVAWAKRCLERESVAKALPESEKVITFISERRKKLG
LE


>gi|2462929|emb|CAA72973.1| Glutathione S-transferase PHI [Arabidopsis thaliana]
MVLKVYGPHFASPKRALVTLIEKGVAFETIPVDLMKGEHKQPAYLALQPFGTVPAVVDGDYKIFESRAVMRYVAEKYRSQGPDLLGKTVEDRGQVEQWLDVEATTYHP
PLLNLTLHIMFASVMGFPSDEKLIKESEEKLAGVLDVYKAQRAKSKYLAGDFVSLADLAHLPFTDYLVGPIGKAYMIKDRKHVSAWWDDISSRPAWKETVAKYSFPA


>gi|732168243|emb|CEG98310.1| Glutathione S-transferase OMEGA [Propionibacterium freudenreichii]
MSTKSTTVGFQLAAPEASHIATSENAHEIGAHGEFSRQDNAFTTPFGDGPGQLPVEAGRYRLIVARICPWAHRQLITREVLGLTDAISVGVTAPVRTDNGWRFSLDPG
DKDPVLGIEYLNEAYLAADPGYDRRGTVPAVVDVTTGKVVQNDYHRLSNYWEVAWRRLQPDDAPDLYPSSLRPAIDEMSRLNFHAVNNGVYKAGFAHTQEAYEQAFDA
LFARLDALEQLLGQQRFLLGEHITDSDVRLFPTLVRFDTAYYVAFRTNRNRLIDFPNLWNYARELYALPGWGSTTDFQAIKLGYFGSTNVTGAARVIIPKGPDLSGWD
QASTRAEQFGDGDVYLRH


The roles of cytosolic GSTs in specific reactions. Each reaction involves nucleophilic attack of GSH upon an electrophilic substrate. A) Synthesis of prostaglandin D2 from prostaglandin H2; B) Isomerization of maleylacetoacetate to fumarylacetoacetate; C) dehalogenation of tetrachlorohydroquinone; D) isomerization of delta-5-androstene-3,17-dione to delta-4-androstene-3,17-dione. [ 1, 2, 3, 4 ].

B1b. Mitochondrial glutathione S-transferase:

>gi|219842317|ref|NP_001137151.1| Glutathione S-transferase KAPPA (mitochondrial GST) [Homo sapiens]
MGPLPRTVELFYDVLSPYSWLGFEILCRYQNIWNINLQLRPSLITGIMKDSGNKPPGLLPRKGLYMANDLKLLRHHLQIPIHFPKDFLSVMLEKGSLSAMRFLTAVNL
EHPEMLEKASRELWMRVWSRVSVGLWESSGRTLDDFLTFPRHVFRVMILPPPGGSTVLPVTPLSPHRLPAVFSSSQNEDITEPQSILAAAEKAGMSAEQAQGLLEKIA
TPKVKNQLKETTEAACRYGAFGLPITVAHVDGQTHMLFGSDRMELLAHLLGEKWMGPIPPAVNARL


B1c. MAPEG superfamily glutathione S-transferase:

>gi|14318725|gb|AAH09155.1| Microsomal glutathione S-transferase (MAPEG superfamily) [Mus musculus]
MADLRQLMDNEVLMAFTSYATIILTKMMFMSSATAFQRITNKVFANPEDCAGFGKVENAKKFVRTDEKVERVRRAHLNDLENIVPFLGIGLLYSLSGPDLSTALMHFR
IFVGARIYHTIAYLTPLPQPNRGLAFFVGYGVTLSMAYRLLRSRLYL


The roles of MAPEG superfamily GSTs in: A) Leukotriene C4 synthase; B) prostaglandin E2 synthase reactions [ 1, 2, 21, 22 ].

Glutathione S-transferase and detoxification reactions.

GSTs play an important role in detoxification of harmful electrophilic compounds in mammalian cells. The liver is the primary site for such detoxification reactions cytosolic GSTs comprise up to 10% of the soluble protein in liver cells. Many harmful compounds, including carcinogens, pesticides, and drugs, are attacked in the liver by cytochrome P450 enzymes in what is referred to as Phase 1 of detoxification. Oxidation of such compounds results in addition of a functional group that can be attacked by Phase II enzymes, which catalyze conjugation of highly polar groups to the newly added functional groups. GSTs are the most important of the Phase II enzymes [ 1, 2, 3, 4, 5, 6, 7 ]. The range of substrates that can be dealt with is expanded by the presence of multiple isozymes of some classes of GSTs. Humans have five genes for Mu class GSTs, four for alpha class GSTs, and two for the theta and omega class GSTs [ 1, 2, 3 ].

The role of GSTs in detoxification reactions [ 1, 2, 5, 7 ].

Glutathione S-transferase and oxidative stress protection [ 1, 2 ].

Similarly to thiol peroxidases (GPXs and PRXs), many GSTs are reduce peroxides. Two molecules of GSH are oxidized to GSSG in this process. Omega class of GSTs is involved in reduction of dehydroascorbate, the oxidized form of vitamin C. Such GSTs have both thioltransferase and dehydroas corbate reductase activity, but little or no activity with electrophilic substrates [ 1, 2, 18 ].

Reduction of cumene hydroperoxide by GSTs [ 1, 2 ].

The major assay for glutathione S-transferase activity measurement [ 23, 24, 25 ].


The glutathione S-transferase assay utilizes 1-Chloro-2,4-dinitrobenzene (CDNB) which can be used as substrate for the broadest range of GSTs. The thiol group of glutathione is conjugate to the CDNB substrate in presanse of GST. The conjugation is accompanied by an increase in absorbance at 340 nm. The rate of increase is directly proportional to the GST activity in the sample [ 23 ].

GSH + CDNB + GST → GS-DNB Conjugate + HCl ;


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