Identification of a binding site for quaternary amines in factor Xa

Citation
D. Monnaie et al., Identification of a binding site for quaternary amines in factor Xa, BIOCHEM, 39(18), 2000, pp. 5349-5354
Citations number
20
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHEMISTRY
ISSN journal
0006-2960 → ACNP
Volume
39
Issue
18
Year of publication
2000
Pages
5349 - 5354
Database
ISI
SICI code
0006-2960(20000509)39:18<5349:IOABSF>2.0.ZU;2-D
Abstract
In the process of characterizing the Naf-binding properties of factor Xa, a specific inhibition of this enzyme by quaternary amines was identified, co nsistent with previous observations. The binding occurs with K-i in the low millimolar range, with trimethylphenylammonium (TMPA) showing the highest specificity. Binding of TMPA inhibits substrate hydrolysis in a competitive manner, does not inhibit the binding of p-aminobenzamidine to the S1 pocke t, and is positively linked to Na+ binding. Inhibition by TMPA is also seen in thrombin and tissue plasminogen activator (tPA), though to a lesser ext ent compared to factor Xa. Computer modeling using the crystal structure of factor Xa suggests that TMPA binds to the S2/S3 specificity sites, with it s hydrophobic moiety making van der Waals interactions with the side chains of Y99, F174, and W215, and the charged amine coupling electrostatically w ith the carboxylates of E97. Site-directed mutagenesis of factor Xa, thromb in, and tPA confirms the predictions drawn by docking calculations and reve al a dominant role for residue Y99. Binding of TMPA to factor Xa is drastic ally (25-fold) reduced by the Y99T replacement. Likewise, the Y99L substitu tion compromises binding of TMPA to tPA. On the other hand, the affinity of TMPA is enhanced 4-fold in thrombin with the substitution L99Y. The identi fication of a binding site for quaternary amines in factor Xa has a bearing on the rational design of selective inhibitors of this clotting enzyme.