This study was designed to identify functionally important factor IX (FIX) residues. Using recombinant techniques and cell culture, we produced a mutant FIX with arginine at 338 changed to alanine (R338A-FIX). This molecule had approximately 3 times greater clotting activity than that of wild type FIX (wt-FIX) in the activated partial thromboplastin assay. R338A-FIX reacted normally with a panel of three FIX specific monoclonal antibodies and migrated on sodium dodecyl sulfate-polyacrylamide gels indistinguishably from wt-FIX. Using functional assays, we determined that R338A-FIXa's Kd for factor VIIIa (FVIIIa) was similar to that of wt-FIXa. Our kinetic analysis, using factor X as substrate, indicated that the mutation's major effects were a 3-fold increase in kcat and a 2-fold decrease in Km both manifested only in the presence of FVIIIa. R338A-FIXa's increased catalytic efficiency did not result from ablation of a thrombin sensitive site, reported to occur at arginine 338, since in our assays the thrombin inhibitor, hirudin, had no effect on activity of either wt-FIXa or R338A-FIXa. R338A-FIXa and wt-FIXa had equal activity, with or without FVIIIa, toward the synthetic substrate, methylsulfonyl-D-cyclohexylglycyl-arginine-p-nitroanilide. Interestingly, R338A-FIXa had reduced affinity for heparin. Therefore, we propose that R338A-FIXa's increased activity is not due to an allosteric effect on the active site, but that the Arg-338 residue is part of an exosite that binds both factor X and the mucopolysaccharide, heparin.
|Seiten (von - bis)||12089-94|
|Fachzeitschrift||The Journal of Biological Chemistry|
|Publikationsstatus||Veröffentlicht - 15 Mai 1998|
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