Molecular Structure at 1·8 Å of Mouse Liver Class Pi Glutathione S-transferase Complexed with S-(p-Nitrobenzyl)glutathione and Other Inhibitors

Garcia-Saez, I.; Parraga, A.; Phillips, Mike; Mantle, Timothy J.; Coll, Miquel

doi:10.1006/jmbi.1994.1232

Cited by 124 publications

(67 citation statements)

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“…Motif I1 is located far from the active site of GST (Figs. 4, 5); recent structural studies did not implicate any of the residues in this motif in binding of either of the substrates (Garcia-Saez et al, 1994;Ji et al, 1994). Rather, these experiments have shown that, unlike the GSHbinding site, the electrophile-binding site is formed by variable segments of the GSTs.…”

Section: Lqkmpvfvgkdg-----fplsetlaiafylas Lgkvpafesadg-----hciaesnaiamentioning

confidence: 98%

Eukaryotic translation elongation factor 1γ contains a glutathione transferase domain—Study of a diverse, ancient protein super family using motif search and structural modeling

et al. 1994

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Using computer methods for multiple alignment, sequence motif search, and tertiary structure modeling, we show that eukaryotic translation elongation factor ly (EFly) contains an N-terminal domain related to class 0 glutathione S-transferases (GST). GST-like proteins related to class 8 comprise a large group including, in addition to typical GSTs and EFly, stress-induced proteins from bacteria and plants, bacterial reductive dehalogenases and P-etherases, and several uncharacterized proteins. These proteins share 2 conserved sequence motifs with GSTs of other classes (a, p, and K). Tertiary structure modeling showed that in spite of the relatively low sequence similarity, the GST-related domain of EFly is likely to form a fold very similar to that in the known structures of class a , p , and ?r GSTs. One of the conserved motifs is implicated in glutathione binding, whereas the other motif probably is involved in maintaining the proper conformation of the GST domain. We predict that the GSTlike domain in EFly is enzymatically active and that to exhibit GST activity, EFly has to form homodimers. The GST activity may be involved in the regulation of the assembly of multisubunit complexes containing EFl and aminoacyl-tRNA synthetases by shifting the balance between glutathione, disulfide glutathione, thiol groups of cysteines, and protein disulfide bonds. The GST domain is a widespread, conserved enzymatic module that may be covalently or noncovalently complexed with other proteins. Regulation of protein assembly and folding may be 1 of the functions of GST.

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Section: Lqkmpvfvgkdg-----fplsetlaiafylas Lgkvpafesadg-----hciaesnaiamentioning

confidence: 98%

Eukaryotic translation elongation factor 1γ contains a glutathione transferase domain—Study of a diverse, ancient protein super family using motif search and structural modeling

et al. 1994

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“…structures of representatives of the a class (Sinning et al, 1993), p class Raghunathan et al, 1994), T class (Reinemer et al, 1991(Reinemer et al, , 1992Garcia-Saez et al, 1994), and u class (Ji et al, 1995) have been determined from X-ray crystallography. The tyrosine residue near the N-terminal end of the enzyme is conserved in all known mammalian cytosolic glutathione S-transferases and is considered to be essential for catalysis (Wilce & Parker, 1994).…”

Section: -1mentioning

confidence: 99%

“…Results of X-ray crystallographic studies on several glutathione S-transferases (Reinemer et al, 1991(Reinemer et al, , 1992Ji et al, 1992;Sinning et al, 1993;Garcia-Saez et al, 1994;Raghunathan et al, 1994) have shown that Tyr 8 is spatially located in the vicinity of the thiol group of the enzyme-bound glutathione. These studies have led to the postulate that this tyrosine residue is essential for the enzymatic mechanism by stabilizing the thiolate anion of the enzymebound glutathione, although it has been noted that positively charged Arg is nearby and may also contribute to stabilizing the thiolate (Stenberg et al, 1991a;Sinning et al, 1993;Wang et al, 1993).…”

Section: Comparison Of Kinetic Constants Of Native and Modified Enzymesmentioning

confidence: 99%

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

1996

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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 I-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, of 0.78 mM and k,,, of 0.01 1 min". 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,S3H]-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,, which has been interpreted as representing the ionization of enzyme-bound glutathione; however, this pK, 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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“…Several amino acid residues of the Pi-class forms including GST P1-1 have been identified in the G-site [9,11,[13][14][15][16]41], but none of them is contained in the C-terminal region between Tyr-198 and Lys-208 recognized by the antibody. Thus it is unlikely that GSH directly binds to the region, thereby inhibiting the interaction with the antibody.…”

Section: Positionmentioning

confidence: 99%

Epitope mapping of a monoclonal antibody to human glutathione transferase P1–1 the binding of which is inhibited by glutathione

Takahata

Tsuchida

Oomura³

et al. 1997

Biochemical Journal

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Although the three-dimensional structure of human glutathione transferase (GST) P1-1 crystallized with a GSH analogue has been reported, its structure in the non-complexed form has not been determined. Four monoclonal antibodies to GST P1-1 were produced to facilitate structural analysis. Of these, one, clone d-1 of IgG #a isotype, dose-dependently inhibited the activity of GST P1-1 but did not affect the activities of either GST A1-1 or M1-1. On immunoblotting, the antibody reacted strongly with GST P1-1 and weakly with rat GST-P and mouse GST-II, indicating cross-reactivity with Pi-class forms but preferential reactivity with GST P1-1. When GST P1-1 and the antibody were incubated in the presence of 60 µM GSH, no inhibition of activity was found, whereas 1-chloro-2,4-dinitrobenzene had no effect at concentrations up to 10 µM. The binding of GST P1-1

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Molecular Structure at 1·8 Å of Mouse Liver Class Pi Glutathione S-transferase Complexed with S-(p-Nitrobenzyl)glutathione and Other Inhibitors

Cited by 124 publications

References 0 publications

Eukaryotic translation elongation factor 1γ contains a glutathione transferase domain—Study of a diverse, ancient protein super family using motif search and structural modeling

Eukaryotic translation elongation factor 1γ contains a glutathione transferase domain—Study of a diverse, ancient protein super family using motif search and structural modeling

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

Epitope mapping of a monoclonal antibody to human glutathione transferase P1–1 the binding of which is inhibited by glutathione

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