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), Al-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 precomplex [k1 ≥ (2-5..