4GSS

HUMAN GLUTATHIONE S-TRANSFERASE P1-1 Y108F MUTANT

4GSS の概要

• エントリーDOI: 10.2210/pdb4gss/pdb
• 分子名称: GLUTATHIONE S-TRANSFERASE, S-HEXYLGLUTATHIONE, 2-(N-MORPHOLINO)-ETHANESULFONIC ACID, ... (4 entities in total)
• 機能のキーワード: transferase, multigene family
• 由来する生物種: Homo sapiens (human)
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 47636.60

構造登録者

Oakley, A.,Rossjohn, J.,Parker, M. (登録日: 1997-01-20, 公開日: 1998-01-28, 最終更新日: 2024-05-22)

主引用文献

Lo Bello, M.,Oakley, A.J.,Battistoni, A.,Mazzetti, A.P.,Nuccetelli, M.,Mazzarese, G.,Rossjohn, J.,Parker, M.W.,Ricci, G.
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, 1997

PubMed 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.

PubMed: 9166793
DOI: 10.1021/bi962813z

実験手法

X-RAY DIFFRACTION (2.5 Å)