Multifunctional Role of Tyr 108 in the Catalytic Mechanism of Human Glutathione Transferase P1-1. Crystallographic and Kinetic Studies on the Y108F Mutant Enzyme

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• 作者：Mario Lo Bello ; Aaron J. Oakley ; Andrea Battistoni ; Anna P. Mazzetti ; Marzia Nuccetelli ; Giampiero Mazzarese ; Jamie Rossjohn ; Michael W. Parker ; and Giorgio Ricci
• 刊名：Biochemistry
• 出版年：1997
• 出版时间：May 20, 1997
• 年：1997
• 卷：36
• 期：20
• 页码：6207 - 6217
• 全文大小：239K
• 年卷期：v.36,no.20(May 20, 1997)
• ISSN：1520-4995

文摘

The possible role of the hydroxyl group of Tyr 108 inthe catalytic mechanism of humanglutathione transferase P1-1 has been investigated by means ofsite-directed mutagenesis, steady-statekinetic analysis, and crystallographic studies. Threerepresentative cosubstrates have been used, i.e.ethacrynic acid, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, and1-chloro-2,4-dinitrobenzene. In the presenceof ethacrynic acid, the enzyme follows a rapid equilibrium randombi-bi mechanism with a rate-limitingstep which occurs after the addition of the substrates and before therelease of products. The replacementof Tyr 108 with Phe yields a 14-fold decrease ofk_cat, while it does not change appreciably theaffinity ofthe H site for the substrate. In this case, it would appear thatthe role of the hydroxyl function is tostabilize the transition state for the chemical step, i.e. the Michaeladdition of GSH to the electrophilicsubstrate. Crystallographic data are compatible with thisconclusion showing the hydroxyl group of Y108in hydrogen bonding distance of the ketone moiety of ethacrynic acid[Oakley, A. J., Rossjohn, J., LoBello, 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 thewild type, suggesting that theremoval of the hydroxyl group is solely responsible for the loss ofactivity. A different involvement ofTyr 108 appears in the catalyzed conjugation of7-chloro-4-nitrobenz-2-oxa-1,3-diazole with GSH in whichthe rate-limiting step is of a physical nature, probably a structuraltransition of the ternary complex. Thesubstitution of Tyr 108 yields an approximately 7-fold increase ofk_cat and a constantk_cat/journals/bichaw/36/i20/eqn/bi962813ze10001.gif">value.Lack of a critical hydrogen bond between7-chloro-4-nitrobenz-2-oxa-1,3-diazole and Tyr 108 appearstobe the basis of the increased k_cat. In the1-chloro-2,4-dinitrobenzene/GSH system, no appreciablechangesof kinetics parameters are found in the Y108F mutant. We concludethat Y108 has a multifunctional rolein glutathione transferase P1-1 catalysis, depending on the nature ofthe electrophilic cosubstrate.