title: Identification of a binding site for quaternary amines in factor Xa.
abstract: In the process of characterizing the Na(+)-binding properties of factor Xa, a specific inhibition of this enzyme by quaternary amines was identified, consistent 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 pocket, and is positively linked to Na(+) binding. Inhibition by TMPA is also seen in thrombin and tissue plasminogen activator (tPA), though to a lesser extent compared to factor Xa. Computer modeling using the crystal structure of factor Xa suggests that TMPA binds to the S2/S3 specificity sites, with its hydrophobic moiety making van der Waals interactions with the side chains of Y99, F174, and W215, and the charged amine coupling electrostatically with the carboxylates of E97. Site-directed mutagenesis of factor Xa, thrombin, and tPA confirms the predictions drawn by docking calculations and reveal a dominant role for residue Y99. Binding of TMPA to factor Xa is drastically (25-fold) reduced by the Y99T replacement. Likewise, the Y99L substitution 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 identification of a binding site for quaternary amines in factor Xa has a bearing on the rational design of selective inhibitors of this clotting enzyme.
authors.affiliation: Cardiovascular Division, Lilly Research Laboratories, Eli Lilly and Co., Indianapolis, Indiana 46285, USA.
authors.forename: D
authors.initials: D
authors.lastname: Monnaie
authors.forename: D
authors.initials: D
authors.lastname: Arosio
authors.forename: N
authors.initials: N
authors.lastname: Griffon
authors.forename: T
authors.initials: T
authors.lastname: Rose
authors.forename: A R
authors.initials: AR
authors.lastname: Rezaie
authors.forename: E
authors.initials: E
authors.lastname: Di Cera
mesh_terms: D000588:Amines / Q000737:chemistry* / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002414:Cations, Monovalent / Q000494:pharmacology; D002863:Chromogenic Compounds / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry; D015951:Factor Xa / Q000737:chemistry* / Q000235:genetics; D065427:Factor Xa Inhibitors; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D011994:Recombinant Proteins; D012964:Sodium / Q000378:metabolism; D013917:Thrombin / Q000737:chemistry / Q000235:genetics; D010959:Tissue Plasminogen Activator / Q000737:chemistry / Q000235:genetics
chemical_list: D000588:Amines; D002414:Cations, Monovalent; D002863:Chromogenic Compounds; D004791:Enzyme Inhibitors; D065427:Factor Xa Inhibitors; D000644:Quaternary Ammonium Compounds; D011994:Recombinant Proteins; C009571:phenyltrimethylammonium; D012964:Sodium; D013917:Thrombin; D015951:Factor Xa; D010959:Tissue Plasminogen Activator