The role of tyrosine-9 and the C-terminal helix in the catalytic mechanism of Alpha-class glutathione S-transferases

Abstract

Glutathione S-transferases (GSTs) play a key role in the metabolism of drugs and xenobiotics. To investigate the catalytic mechanism, substrate binding and catalysis by the wild-type and two mutants of GST A1-1 have been studied. Substitution of the 'essential' Tyr(9) by phenylalanine leads to a marked decrease in the k(cat) for 1-chloro-2,4-dinitrobenzene (CDNB), but has no affect on k(cat) for ethacrynic acid. Similarly, removal of the C-terminal helix by truncation of the enzyme at residue 209 leads to a decrease in k(cat) for CDNB, but an increase in k(cat) for ethacrynic acid. The binding of a GSH analogue increases the affinity of the wild-type enzyme for CDNB, and increases the rate of the enzyme-catalysed conjugation of this substrate with the small thiols 2-mercaptoethanol and dithiothreitol. This suggests that GSH binding produces a conformational change which is transmitted to the binding site for the hydrophobic substrate, where it alters both the affinity for the substrate and the catalytic-centre activity ('turnover number') for conjugation, perhaps by increasing the proportion of the substrate bound productively. Neither of these two effects of GSH analogues are seen in the C-terminally truncated enzyme, indicating a role for the C-terminal helix in the GSH-induced conformational change.

The Catalytic Tyr-9 of Glutathione S-Transferase A1-1 Controls the Dynamics of the C terminus

“…The k Ϫ2(exp) could not be determined directly, presumably because GS-EA binds too tightly and is not displaced by the trapping agent, GSO 3 Ϫ . Surprisingly, in steady state reactions with hGST A1-1 and EA, k cat was unchanged upon substitution at Tyr-9, in contrast to our results, which suggest a decreased k cat for a Y9F-catalyzed reaction, which is rate-limited by product release (24). The source of these differences is unknown.…”

“…A similar effect was observed in equilibrium binding studies with Y9F hGST A1-1. Allardyce et al (24) recently found that Y9F hGST A1-1 binds the ligand, S-dinitrophenylglutathione, 20-fold more tightly than WT. ⌬H°and ⌬S°values also indicate a difference in the thermodynamics of binding to each enzyme.…”

“…Although it is unclear whether the residues are completely disordered or lie in a delocalized helix, the C terminus will be referred to here as "disordered" in this unobservable state. In conjunction, the "folded" or "ordered" state refers to the observable helix when a GS-product conjugate is bound.Previously, contributions of the helix to catalysis, ligandin function, and ligand dissociation and binding in GST A1-1 have been determined (24,(27)(28)(29)(30)(31). However, the detailed mechanism for, and the residues involved in, the ordering of the helix have not been elucidated.…”

“…Previously, contributions of the helix to catalysis, ligandin function, and ligand dissociation and binding in GST A1-1 have been determined (24,(27)(28)(29)(30)(31). However, the detailed mechanism for, and the residues involved in, the ordering of the helix have not been elucidated.…”

An Electron-sharing Network Involved in the Catalytic Mechanism Is Functionally Conserved in Different Glutathione Transferase Classes

“…Many of the mechanistic investigations of GSTs have emphasized the importance of a conserved tyrosine or serine residue at the glutathione binding site of certain GSTs (Tyr-8 for Pi, Tyr-9 for Alpha, Tyr-6 for Mu, and Ser-9 for Delta class) in facilitating the thiol deprotonation (10,(21)(22)(23). The hydroxyl group of this tyrosine or serine residue that is within hydrogenbonding distance of the thiol group of enzyme-bound glutathione is considered to be required for the correct orientation and stabilization of the deprotonated thiolate anion in the active site (10, 22, 24 -26).…”