Identification of a binding site for quaternary amines in factor Xa

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.