Changing residue 338 in human factor IX from arginine to alanine causes an increase in catalytic activity.
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.
Chang
J
J
Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599-3280, USA.
Jin
J
J
Lollar
P
P
Bode
W
W
Brandstetter
H
H
Hamaguchi
N
N
Straight
D L
DL
Stafford
D W
DW
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
D006629:Hirudins; D011994:Recombinant Proteins; D015944:Factor VIIIa; D005164:Factor IX; D006493:Heparin; D001120:Arginine; D000409:Alanine