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

H. Cheng, T. Tchaikovskaya, Y. S.L. Tu, J. Chapman, B. Qian, W. M. Ching, M. Tien, J. D. Rowe, Y. V. Patskovsky, I. Listowsky, C. P.D. Tu

Research output: Contribution to journalArticle

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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 (kcat/Km) 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 α4 region of rGSTM4-4 account for this pKa 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.

Original languageEnglish (US)
Pages (from-to)403-414
Number of pages12
JournalBiochemical Journal
Volume356
Issue number2
DOIs
StatePublished - Jun 1 2001

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Glutathione Disulfide
Glutathione Transferase
Disulfides
Isoenzymes
Oxidation-Reduction
Glutathione
Cysteine
Rats
Liver
Genes
Dinitrochlorobenzene
Oxidative stress
Molecular modeling
Enzyme activity
Enzymes
Sulfhydryl Compounds
Escherichia coli
Rodentia
Oxidative Stress
Substitution reactions

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Cheng, H. ; Tchaikovskaya, T. ; Tu, Y. S.L. ; Chapman, J. ; Qian, B. ; Ching, W. M. ; Tien, M. ; Rowe, J. D. ; Patskovsky, Y. V. ; Listowsky, I. ; Tu, C. P.D. / 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. In: Biochemical Journal. 2001 ; Vol. 356, No. 2. pp. 403-414.
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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 (kcat/Km) 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 α4 region of rGSTM4-4 account for this pKa 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.",
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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. / Cheng, H.; Tchaikovskaya, T.; Tu, Y. S.L.; Chapman, J.; Qian, B.; Ching, W. M.; Tien, M.; Rowe, J. D.; Patskovsky, Y. V.; Listowsky, I.; Tu, C. P.D.

In: Biochemical Journal, Vol. 356, No. 2, 01.06.2001, p. 403-414.

Research output: Contribution to journalArticle

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T1 - Rat glutathione S-transferase M4-4

T2 - An isoenzyme with unique structural features including a redox-reactive cysteine-115 residue that forms mixed disulphides with glutathione

AU - Cheng, H.

AU - Tchaikovskaya, T.

AU - Tu, Y. S.L.

AU - Chapman, J.

AU - Qian, B.

AU - Ching, W. M.

AU - Tien, M.

AU - Rowe, J. D.

AU - Patskovsky, Y. V.

AU - Listowsky, I.

AU - Tu, C. P.D.

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N2 - 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 (kcat/Km) 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 α4 region of rGSTM4-4 account for this pKa 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.

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