PDBsum entry 4gss

Transferase

PDB id: 4gss

Contents

Protein chain

208 a.a. *

Ligands

GTX ×2

MES ×2

Waters ×189

* Residue conservation analysis

PDB id:

4gss

Name:

Transferase

Title:

Human glutathione s-transferase p1-1 y108f mutant

Structure:

Glutathione s-transferase. Chain: a, b. Engineered: yes. Mutation: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Organ: placenta. Gene: gtp_human. Expressed in: escherichia coli. Expression_system_taxid: 562

Biol. unit:

Dimer (from PQS)

Resolution:

2.50Å R-factor: 0.198 R-free: 0.262

Authors:

A.Oakley,J.Rossjohn,M.Parker

Key ref:

M.Lo Bello et al. (1997). Multifunctional role of Tyr 108 in the catalytic mechanism of human glutathione transferase P1-1. Crystallographic and kinetic studies on the Y108F mutant enzyme. Biochemistry, 36, 6207-6217. PubMed id: 9166793 DOI: 10.1021/bi962813z

Date:

20-Jan-97 Release date: 28-Jan-98

Protein chains

P09211  (GSTP1_HUMAN) -  Glutathione S-transferase P from Homo sapiens

Seq: 210 a.a.

Struc: 208 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.2.5.1.18 - glutathione transferase.
• Reaction: RX + glutathione = an S-substituted glutathione + a halide anion + H⁺

RX (Bound ligand (Het Group name = GTX) matches with 76.92% similarity) + glutathione = S-substituted glutathione + halide anion + H(+)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

Added reference

DOI no: 10.1021/bi962813z

Biochemistry 36:6207-6217 (1997)

PubMed id: 9166793

Multifunctional role of Tyr 108 in the catalytic mechanism of human glutathione transferase P1-1. Crystallographic and kinetic studies on the Y108F mutant enzyme.

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

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.