Valine 10 May Act as a Driver for Product Release from the Active Site of Human Glutathione Transferase P1-1

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• 作者：Chiara Micaloni ; Anna P. Mazzetti ; Marzia Nuccetelli ; Jamie Rossjohn ; William J. McKinstry ; Giovanni Antonini ; Anna M. Caccuri ; Aaron J. Oakley ; Giorgio Federici ; Giorgio Ricci ; Michael W. Parker ; and
• 刊名：Biochemistry
• 出版年：2000
• 出版时间：December 26, 2000
• 年：2000
• 卷：39
• 期：51
• 页码：15961 - 15970
• 全文大小：136K
• 年卷期：v.39,no.51(December 26, 2000)
• ISSN：1520-4995

文摘

We have probed the electrophilic binding site (H-site) of human glutathione transferase P1-1through mutagenesis of two valines, Val 10 and Val 35, into glycine and alanine, respectively. These tworesidues were previously shown to be the only conformationally variable residues in the H-site and hencemay play important roles in cosubstrate recognition and/or product dissociation. Both of these mutantenzymes have been expressed in Escherichia coli and purified and their kinetic properties characterized.The results demonstrate that Val35Ala behaves similarly to wild-type, whereas Val10Gly exhibits a strongdecrease of k_cat and k_cat/K_m ^cosub toward two selected cosubstrates: ethacrynic acid and 1-chloro-2,4-dinitrobenzene. Pre-steady-state kinetic analysis of the GSH conjugation with ethacrynic acid shows thatboth wild-type and Val10Gly mutant enzymes exhibit the same rate-limiting step: the dissociation ofproduct. However, in the Val10Gly mutant there is an increased energetic barrier which renders thedissociation of product more difficult. Similar results are found for the Val10Gly mutant with 1-chloro-2,4-dinitrobenzene as cosubstrate. With this latter cosubstrate, Val 10 also exerts a positive role in theconformational transitions of the ternary complex before the chemical event. Crystallographic analysis ofthe Val10Gly mutant in complex with the inhibitor S-hexyl-GSH suggests that Val 10 optimally orientatesproducts, thus promoting their exit from the active site.