title: Proton release on binding of glutathione to alpha, Mu and Delta class glutathione transferases.
abstract: Potentiometric, spectroscopic and stopped-flow experiments have been performed to dissect the binding mechanism of GSH to selected glutathione S-transferases (GSTs), A1-1, M2-2 and Lucilia cuprina GST, belonging to Alpha, Mu and Delta classes respectively. Both Alpha and Mu isoenzymes quantitatively release the thiol proton of the substrate when the binary complex is formed. Proton extrusion, quenching of intrinsic fluorescence and thiolate formation, diagnostic of different steps along the binding pathway, have been monitored by stopped-flow analysis. Kinetic data are consistent with a multi-step binding mechanism: the substrate is initially bound to form an un-ionized pre-complex [k(1)>/=(2-5)x10(6) M(-1).s(-1)], which is slowly converted into the final Michaelis complex (k(2)=1100-1200 s(-1)). Ionization of GSH, fluorescence quenching and proton extrusion are fast events that occur either synchronously or rapidly after the final complex formation. The Delta isoenzyme shows an interesting difference: proton extrusion is almost stoichiometric with thiolate formed at the active site only up to pH 7.0. Above this pH, at least one protein residue acts as internal base to neutralize the thiol proton. These results suggest that the Alpha and Mu enzymes retain not only a similar catalytic outcome and overall three-dimensional structure but also share a similar kinetic mechanism for GSH binding. The Delta GST, which is closely related to the mammalian Theta class enzymes and is distantly related to Alpha and Mu GSTs in the evolutionary pathway, might display a different activation mechanism for GSH.
authors.affiliation: Department of Biology, University of Rome 'Tor Vergata', via della Ricerca Scientifica 00133 Rome, Italy.
authors.forename: A M
authors.initials: AM
authors.lastname: Caccuri
authors.forename: G
authors.initials: G
authors.lastname: Antonini
authors.forename: P G
authors.initials: PG
authors.lastname: Board
authors.forename: M W
authors.initials: MW
authors.lastname: Parker
authors.forename: M
authors.initials: M
authors.lastname: Nicotra
authors.forename: M
authors.initials: M
authors.lastname: Lo Bello
authors.forename: G
authors.initials: G
authors.lastname: Federici
authors.forename: G
authors.initials: G
authors.lastname: Ricci
mesh_terms: D000818:Animals; D004175:Diptera; D017022:Flow Injection Analysis; D005978:Glutathione / Q000378:metabolism*; D005982:Glutathione Transferase / Q000145:classification / Q000378:metabolism*; D006801:Humans; D007477:Ions; D007527:Isoenzymes / Q000145:classification / Q000378:metabolism*; D008956:Models, Chemical; D011199:Potentiometry; D011522:Protons; D013050:Spectrometry, Fluorescence
chemical_list: D007477:Ions; D007527:Isoenzymes; D011522:Protons; D005982:Glutathione Transferase; D005978:Glutathione