Tyrosine 8 contributes to catalysis but is not required for activity of rat liver glutathione S-transferase, 1-1

Jibo Wang, Joseph J. Barycki, Roberta F. Colman

Research output: Contribution to journalArticle

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Abstract

Reaction of rat liver glutathione S-transferase, isozyme 1-1, with 4- (fluorosulfonyl)benzoic acid (4-FSB), a xenobiotic substrate analogue, results in a time-dependent inactivation of the enzyme to a final value of 35% of its original activity when assayed at pH 6.5 with 1-chloro-2,4- dinitrobenzene (CDNB) as substrate. The rate of inactivation exhibits a nonlinear dependence on the concentration of 4-FSB from 0.25 mM to 9 mM, characterized by a K(l) of 0,78 mM and k(max) of 0.011 min-1. S- Hexylglutathione or the xenobiotic substrate analogue, 2,4-dinitrophenol, protects against inactivation of the enzyme by 4-FSB, whereas S- methylglutathione has little effect on the reaction. These experiments indicate that reaction occurs within the active site of the enzyme, probably in the binding site of the xenobiotic substrate, close to the glutathione binding site. Incorporation of [3,5-3H]-4-FSB into the enzyme in the absence and presence of S-hexylglutathione suggests that modification of one residue is responsible for the partial loss of enzyme activity. Tyr 8 and Cys 17 are shown to be the reaction targets of 4-FSB, but only Tyr 8 is protected against 4-FSB by S-hexylglutathione. DTT regenerates cysteine from the reaction product of cysteine and 4-FSB, but does not reactivate the enzyme. These results show that modification of Tyr 8 by 4-FSB causes the partial inactivation of the enzyme. The Michaelis constants for various substrates are not changed by the modification of the enzyme. The pH dependence of the enzyme-catalyzed reaction of glutathione with CDNB for the modified enzyme, as compared with the native enzyme, reveals an increase of about 0.9 in the apparent pK(a), which has been interpreted as representing the ionization of enzyme-bound glutathione; however, this pK(a) of about 7.4 for modified enzyme remains far below the pK of 9.1 for the -SH of free glutathione. Previously, it was considered that Tyr 8 was essential for GST catalysis. In contrast, we conclude that Tyr 8 facilitates the ionization of the thiol group of glutathione bound to glutathione S-transferase, but is not required for enzyme activity.

Original languageEnglish (US)
Pages (from-to)1032-1042
Number of pages11
JournalProtein Science
Volume5
Issue number6
StatePublished - Jun 1 1996

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Glutathione Transferase
Catalysis
Liver
Tyrosine
Rats
Enzymes
Glutathione
Xenobiotics
Substrates
Enzyme activity
Ionization
Cysteine
Binding Sites
2,4-Dinitrophenol
Dinitrochlorobenzene
4-(fluorosulfonyl)benzoic acid
Reaction products
Sulfhydryl Compounds
Isoenzymes
Catalytic Domain

Keywords

  • 4-(fluorosulfonyl)benzoic acid
  • affinity labeling
  • glutathione S- transferase

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Tyrosine 8 contributes to catalysis but is not required for activity of rat liver glutathione S-transferase, 1-1. / Wang, Jibo; Barycki, Joseph J.; Colman, Roberta F.

In: Protein Science, Vol. 5, No. 6, 01.06.1996, p. 1032-1042.

Research output: Contribution to journalArticle

Wang, Jibo ; Barycki, Joseph J. ; Colman, Roberta F. / Tyrosine 8 contributes to catalysis but is not required for activity of rat liver glutathione S-transferase, 1-1. In: Protein Science. 1996 ; Vol. 5, No. 6. pp. 1032-1042.
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abstract = "Reaction of rat liver glutathione S-transferase, isozyme 1-1, with 4- (fluorosulfonyl)benzoic acid (4-FSB), a xenobiotic substrate analogue, results in a time-dependent inactivation of the enzyme to a final value of 35{\%} of its original activity when assayed at pH 6.5 with 1-chloro-2,4- dinitrobenzene (CDNB) as substrate. The rate of inactivation exhibits a nonlinear dependence on the concentration of 4-FSB from 0.25 mM to 9 mM, characterized by a K(l) of 0,78 mM and k(max) of 0.011 min-1. S- Hexylglutathione or the xenobiotic substrate analogue, 2,4-dinitrophenol, protects against inactivation of the enzyme by 4-FSB, whereas S- methylglutathione has little effect on the reaction. These experiments indicate that reaction occurs within the active site of the enzyme, probably in the binding site of the xenobiotic substrate, close to the glutathione binding site. Incorporation of [3,5-3H]-4-FSB into the enzyme in the absence and presence of S-hexylglutathione suggests that modification of one residue is responsible for the partial loss of enzyme activity. Tyr 8 and Cys 17 are shown to be the reaction targets of 4-FSB, but only Tyr 8 is protected against 4-FSB by S-hexylglutathione. DTT regenerates cysteine from the reaction product of cysteine and 4-FSB, but does not reactivate the enzyme. These results show that modification of Tyr 8 by 4-FSB causes the partial inactivation of the enzyme. The Michaelis constants for various substrates are not changed by the modification of the enzyme. The pH dependence of the enzyme-catalyzed reaction of glutathione with CDNB for the modified enzyme, as compared with the native enzyme, reveals an increase of about 0.9 in the apparent pK(a), which has been interpreted as representing the ionization of enzyme-bound glutathione; however, this pK(a) of about 7.4 for modified enzyme remains far below the pK of 9.1 for the -SH of free glutathione. Previously, it was considered that Tyr 8 was essential for GST catalysis. In contrast, we conclude that Tyr 8 facilitates the ionization of the thiol group of glutathione bound to glutathione S-transferase, but is not required for enzyme activity.",
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N2 - Reaction of rat liver glutathione S-transferase, isozyme 1-1, with 4- (fluorosulfonyl)benzoic acid (4-FSB), a xenobiotic substrate analogue, results in a time-dependent inactivation of the enzyme to a final value of 35% of its original activity when assayed at pH 6.5 with 1-chloro-2,4- dinitrobenzene (CDNB) as substrate. The rate of inactivation exhibits a nonlinear dependence on the concentration of 4-FSB from 0.25 mM to 9 mM, characterized by a K(l) of 0,78 mM and k(max) of 0.011 min-1. S- Hexylglutathione or the xenobiotic substrate analogue, 2,4-dinitrophenol, protects against inactivation of the enzyme by 4-FSB, whereas S- methylglutathione has little effect on the reaction. These experiments indicate that reaction occurs within the active site of the enzyme, probably in the binding site of the xenobiotic substrate, close to the glutathione binding site. Incorporation of [3,5-3H]-4-FSB into the enzyme in the absence and presence of S-hexylglutathione suggests that modification of one residue is responsible for the partial loss of enzyme activity. Tyr 8 and Cys 17 are shown to be the reaction targets of 4-FSB, but only Tyr 8 is protected against 4-FSB by S-hexylglutathione. DTT regenerates cysteine from the reaction product of cysteine and 4-FSB, but does not reactivate the enzyme. These results show that modification of Tyr 8 by 4-FSB causes the partial inactivation of the enzyme. The Michaelis constants for various substrates are not changed by the modification of the enzyme. The pH dependence of the enzyme-catalyzed reaction of glutathione with CDNB for the modified enzyme, as compared with the native enzyme, reveals an increase of about 0.9 in the apparent pK(a), which has been interpreted as representing the ionization of enzyme-bound glutathione; however, this pK(a) of about 7.4 for modified enzyme remains far below the pK of 9.1 for the -SH of free glutathione. Previously, it was considered that Tyr 8 was essential for GST catalysis. In contrast, we conclude that Tyr 8 facilitates the ionization of the thiol group of glutathione bound to glutathione S-transferase, but is not required for enzyme activity.

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KW - 4-(fluorosulfonyl)benzoic acid

KW - affinity labeling

KW - glutathione S- transferase

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