These tools will no longer be maintained as of December 31, 2024. Archived website can be found here. PubMed4Hh GitHub repository can be found here. Contact NLM Customer Service if you have questions.


Pubmed for Handhelds

PUBMED FOR HANDHELDS

Search MEDLINE/PubMed

  • Title: Valine 10 may act as a driver for product release from the active site of human glutathione transferase P1-1.
    Author: Micaloni C, Mazzetti AP, Nuccetelli M, Rossjohn J, McKinstry WJ, Antonini G, Caccuri AM, Oakley AJ, Federici G, Ricci G, Parker MW, Lo Bello M.
    Journal: Biochemistry; 2000 Dec 26; 39(51):15961-70. PubMed ID: 11123923.
    Abstract:
    We have probed the electrophilic binding site (H-site) of human glutathione transferase P1-1 through mutagenesis of two valines, Val 10 and Val 35, into glycine and alanine, respectively. These two residues were previously shown to be the only conformationally variable residues in the H-site and hence may play important roles in cosubstrate recognition and/or product dissociation. Both of these mutant enzymes 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 strong decrease 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 that both wild-type and Val10Gly mutant enzymes exhibit the same rate-limiting step: the dissociation of product. However, in the Val10Gly mutant there is an increased energetic barrier which renders the dissociation 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 the conformational transitions of the ternary complex before the chemical event. Crystallographic analysis of the Val10Gly mutant in complex with the inhibitor S-hexyl-GSH suggests that Val 10 optimally orientates products, thus promoting their exit from the active site.
    [Abstract] [Full Text] [Related] [New Search]