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

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)/K-m(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

Generating Personalized and Certifiable Workflow Designs: A Prototype

International audienceAs the first level of a BPM strategy, being able to design event-oriented models of processes is a must-have competence for every modern business. Unfortunately, industrial procedures have reached a certain complexity making the designing task complex enough to discourage businesses facing the blank page. Moreover, the 21st century witnesses the emergence of myriads of norms and external regulations that businesses want to abide by. Although domain experts have a limited process modelling and norm interpretation knowledge, they know how to describe their activities and their sequencing. With progresses made in the artificial intelligence, particularly in the natural language processing domain, it becomes possible to automatize the task of creating a process in compliance with norms. This paper presents a business-oriented prototype assisting users in getting certifiable specific business processes. We detail the metamodel used to separately model norms and business’ existing procedures and then, the algorithm envisaged to deduce a corresponding cartography of processes