title: Rat glutathione S-transferase M4-4: an isoenzyme with unique structural features including a redox-reactive cysteine-115 residue that forms mixed disulphides with glutathione.
abstract: Although the existence of the rat glutathione S-transferase (GST) M4 (rGSTM4) gene has been known for some time, the corresponding protein has not as yet been purified from tissue. A recombinant rGSTM4-4 was thus expressed in Escherichia coli from a chemically synthesized rGSTM4 gene. The catalytic efficiency (k(cat)/K(m)) of rGSTM4-4 for the 1-chloro-2,4-dinitrobenzene (CDNB) conjugation reaction was 50-180-fold less than that of the well-characterized homologous rGSTM1-1, and the pH optimum for the same reaction was 8.5 for rGSTM4-4 as opposed to 6.5 for rGSTM1-1. Molecular-modelling studies predict that key substitutions in the helix alpha4 region of rGSTM4-4 account for this pK(a) difference. A notable structural feature of rGSTM4-4 is the Cys-115 residue in place of the Tyr-115 of other Mu-class GSTs. The thiol group of Cys-115 is redox-reactive and readily forms a mixed disulphide even with GSH; the S-glutathiolated form of the enzyme is catalytically active. A mutated rGSTM4-4 (C115Y) had 6-10-fold greater catalytic efficiency than the wild-type rGSTM4-4. Trp-45, a conserved residue among Mu-class GSTs, is essential in rGSTM4-4 for both enzyme activity and binding to glutathione affinity matrices. Antibodies directed against either the unique C-terminal undecapeptide or tridecapeptide of rGSTM4 reacted with rat and mouse liver GSTs to reveal an orthologous mouse GSTM4-4 present at low basal levels but which is inducible in mouse liver. This subclass of rodent Mu GSTs with redox-active Cys-115 residues could have specialized physiological functions in response to oxidative stress.
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA.
authors.forename: H
authors.initials: H
authors.lastname: Cheng
authors.forename: T
authors.initials: T
authors.lastname: Tchaikovskaya
authors.forename: Y S
authors.initials: YS
authors.lastname: Tu
authors.forename: J
authors.initials: J
authors.lastname: Chapman
authors.forename: B
authors.initials: B
authors.lastname: Qian
authors.forename: W M
authors.initials: WM
authors.lastname: Ching
authors.forename: M
authors.initials: M
authors.lastname: Tien
authors.forename: J D
authors.initials: JD
authors.lastname: Rowe
authors.forename: Y V
authors.initials: YV
authors.lastname: Patskovsky
authors.forename: I
authors.initials: I
authors.lastname: Listowsky
authors.forename: C P
authors.initials: CP
authors.lastname: Tu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003545:Cysteine / Q000737:chemistry; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D019281:Dimerization; D004220:Disulfides / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D010084:Oxidation-Reduction; D011487:Protein Conformation; D021122:Protein Subunits; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D004220:Disulfides; D007527:Isoenzymes; D021122:Protein Subunits; D011994:Recombinant Proteins; D005982:Glutathione Transferase; D005978:Glutathione; D003545:Cysteine