title: The three-dimensional structure of Cys-47-modified mouse liver glutathione S-transferase P1-1. Carboxymethylation dramatically decreases the affinity for glutathione and is associated with a loss of electron density in the alphaB-310B region.
abstract: The three-dimensional structure of mouse liver glutathione S-transferase P1-1 carboxymethylated at Cys-47 and its complex with S-(p-nitrobenzyl)glutathione have been determined by x-ray diffraction analysis. The structure of the modified enzyme described here is the first structural report for a Pi class glutathione S-transferase with no glutathione, glutathione S-conjugate, or inhibitor bound. It shows that part of the active site area, which includes helix alphaB and helix 310B, is disordered. However, the environment of Tyr-7, an essential residue for the catalytic reaction, remains unchanged. The position of the sulfur atom of glutathione is occupied in the ligand-free enzyme by a water molecule that is at H-bond distance from Tyr-7. We do not find any structural evidence for a tyrosinate form, and therefore our results suggest that Tyr-7 is not acting as a general base abstracting the proton from the thiol group of glutathione. The binding of the inhibitor S-(p-nitrobenzyl)-glutathione to the carboxymethylated enzyme results in a partial restructuring of the disordered area. The modification of Cys-47 sterically hinders structural organization of this region, and although it does not prevent glutathione binding, it significantly reduces the affinity. A detailed kinetic study of the modified enzyme indicates that the carboxymethylation increases the Km for glutathione by 3 orders of magnitude, although the enzyme can function efficiently under saturating conditions.
authors.affiliation: Departament de Biologia Molecular i Cel.lular, Centre d'Investigació i Desenvolupament-Consell Superior d'Investigacions Científiques, Jordi Girona 18-26, 08034 Barcelona, Spain.
authors.forename: M C
authors.initials: MC
authors.lastname: Vega
authors.forename: S B
authors.initials: SB
authors.lastname: Walsh
authors.forename: T J
authors.initials: TJ
authors.lastname: Mantle
authors.forename: M
authors.initials: M
authors.lastname: Coll
mesh_terms: D000818:Animals; D001665:Binding Sites; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000031:analogs & derivatives; D005978:Glutathione / Q000031:analogs & derivatives* / Q000737:chemistry; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D007461:Iodoacetates / Q000737:chemistry; D019807:Iodoacetic Acid; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry*; D008099:Liver / Q000201:enzymology; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation
chemical_list: D007461:Iodoacetates; D007527:Isoenzymes; C038391:S-(4-nitrobenzyl)glutathione; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase; C496557:Gstp1 protein, mouse; D005978:Glutathione; D003545:Cysteine; D019807:Iodoacetic Acid