title: Changing residue 338 in human factor IX from arginine to alanine causes an increase in catalytic activity.
abstract: 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.
authors.affiliation: Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599-3280, USA.
authors.forename: J
authors.initials: J
authors.lastname: Chang
authors.forename: J
authors.initials: J
authors.lastname: Jin
authors.forename: P
authors.initials: P
authors.lastname: Lollar
authors.forename: W
authors.initials: W
authors.lastname: Bode
authors.forename: H
authors.initials: H
authors.lastname: Brandstetter
authors.forename: N
authors.initials: N
authors.lastname: Hamaguchi
authors.forename: D L
authors.initials: DL
authors.lastname: Straight
authors.forename: D W
authors.initials: DW
authors.lastname: Stafford
mesh_terms: D000409:Alanine / Q000737:chemistry / Q000378:metabolism*; D019943:Amino Acid Substitution; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D005164:Factor IX / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D015944:Factor VIIIa / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology; D006629:Hirudins / Q000494:pharmacology; D006801:Humans; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D006629:Hirudins; D011994:Recombinant Proteins; D015944:Factor VIIIa; D005164:Factor IX; D006493:Heparin; D001120:Arginine; D000409:Alanine