PDBsum entry 4gss

Transferase

PDB id: 4gss

Contents

Protein chain

208 a.a. *

Ligands

GTX ×2

MES ×2

Waters ×189

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Multifunctional role of tyr 108 in the catalytic mechanism of human glutathione transferase p1-1. Crystallographic and kinetic studies on the y108f mutant enzyme.

Authors

M.Lo bello, A.J.Oakley, A.Battistoni, A.P.Mazzetti, M.Nuccetelli, G.Mazzarese, J.Rossjohn, M.W.Parker, G.Ricci.

Ref.

Biochemistry, 1997, 36, 6207-6217. [DOI no: 10.1021/bi962813z]

PubMed id

9166793

Abstract

The possible role of the hydroxyl group of Tyr 108 in the catalytic mechanism of human glutathione transferase P1-1 has been investigated by means of site-directed mutagenesis, steady-state kinetic analysis, and crystallographic studies. Three representative cosubstrates have been used, i.e. ethacrynic acid, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, and 1-chloro-2,4-dinitrobenzene. In the presence of ethacrynic acid, the enzyme follows a rapid equilibrium random bi-bi mechanism with a rate-limiting step which occurs after the addition of the substrates and before the release of products. The replacement of Tyr 108 with Phe yields a 14-fold decrease of k(cat), while it does not change appreciably the affinity of the H site for the substrate. In this case, it would appear that the role of the hydroxyl function is to stabilize the transition state for the chemical step, i.e. the Michael addition of GSH to the electrophilic substrate. Crystallographic data are compatible with this conclusion showing the hydroxyl group of Y108 in hydrogen bonding distance of the ketone moiety of ethacrynic acid [Oakley, A. J., Rossjohn, J., Lo Bello, M., Caccuri, A. M., Federici, G., & Parker, M. W. (1997) Biochemistry 36, 576-585]. Moreover, no structural differences are observed between the Y108F mutant and the wild type, suggesting that the removal of the hydroxyl group is solely responsible for the loss of activity. A different involvement of Tyr 108 appears in the catalyzed conjugation of 7-chloro-4-nitrobenz-2-oxa-1,3-diazole with GSH in which the rate-limiting step is of a physical nature, probably a structural transition of the ternary complex. The substitution of Tyr 108 yields an approximately 7-fold increase of k(cat) and a constant k(cat)/Km(NBD-Cl) value. Lack of a critical hydrogen bond between 7-chloro-4-nitrobenz-2-oxa-1,3-diazole and Tyr 108 appears to be the basis of the increased k(cat). In the 1-chloro-2,4-dinitrobenzene/GSH system, no appreciable changes of kinetics parameters are found in the Y108F mutant. We conclude that Y108 has a multifunctional role in glutathione transferase P1-1 catalysis, depending on the nature of the electrophilic cosubstrate.

Secondary reference #1

Title

Three-Dimensional structure of class pi glutathione s-Transferase from human placenta in complex with s-Hexylglutathione at 2.8 a resolution.

Authors

P.Reinemer, H.W.Dirr, R.Ladenstein, R.Huber, M.Lo bello, G.Federici, M.W.Parker.

Ref.

J Mol Biol, 1992, 227, 214-226. [DOI no: 10.1016/0022-2836(92)90692-D]

PubMed id

1522586

Figure 8.
Figure 8. Conolly dot surface of the op region of human class x glutathione S-transferase showing both active sites occupied by S-hexyllutathione. View is along the local dyad. Also shown is the cavity formed between the 2 subunits.

Figure 9.
Figure 9. Model o inhibitor S-hexylglutathione and its next neighbors at the active site of human -transferase.

The above figures are reproduced from the cited reference with permission from Elsevier