Spectroscopic and kinetic evidence for the thiolate anion of glutathione at the active site of glutathione S-transferase
| Autor: | Richard N. Armstrong, Gerard F. Graminski, Yasuo Kubo |
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| Rok vydání: | 1989 |
| Předmět: |
Anions
Stereochemistry Tripeptide In Vitro Techniques Photochemistry Biochemistry chemistry.chemical_compound Animals Carboxylate Binding site Glutathione Transferase chemistry.chemical_classification Binding Sites biology Chemistry Active site Glutathione Hydrogen-Ion Concentration Rats Isoenzymes Dissociation constant Kinetics Liver biology.protein Thiol Spectrophotometry Ultraviolet Titration |
| Zdroj: | Biochemistry. 28:3562-3568 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi00434a062 |
| Popis: | Ultraviolet difference spectroscopy of the binary complex of isozyme 4-4 of rat liver glutathione S-transferase with glutathione (GSH) and the enzyme alone or as the binary complex with the oxygen analogue, gamma-L-glutamyl-L-serylglycine (GOH), at neutral pH reveals an absorption band at 239 nm (epsilon = 5200 M-1 cm-1) that is assigned to the thiolate anion (GS-) of the bound tripeptide. Titration of this difference absorption band over the pH range 5-8 indicates that the thiol of enzyme-bound GSH has a pKa = 6.6, which is about 2.4 pK units less than that in aqueous solution and consistent with the kinetically determined pKa previously reported [Chen et al. (1988) Biochemistry 27, 647]. The observed shift in the pKa between enzyme-bound and free GSH suggests that about 3.3 kcal/mol of the intrinsic binding energy of the peptide is utilized to lower the pKa into the physiological pH range. Apparent dissociation constants for both GSH and GOH are comparable and vary by a factor of less than 2 over the same pH range. Site occupancy data and spectral band intensity reveal large extinction coefficients at 239 nm (epsilon = 5200 M-1 cm-1) and 250 nm (epsilon = 1100 M-1 cm-1) that are consistent with the existence of either a glutathione thiolate (E.GS-) or ion-paired thiolate (EH+.GS-) in the active site. The observation that GS- is likely the predominant tripeptide species bound at the active site suggested that the carboxylate analogue of GSH, gamma-L-glutamyl-(D,L-2-aminomalonyl)glycine, should bind more tightly than GSH.(ABSTRACT TRUNCATED AT 250 WORDS) |
| Databáze: | OpenAIRE |
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