title: An Arg/Ser substitution in the second epidermal growth factor-like module of factor IX introduces an O-linked carbohydrate and markedly impairs activation by factor XIa and factor VIIa/Tissue factor and catalytic efficiency of factor IXa.
abstract: Factor IXR94S is a naturally occurring hemophilia B defect, which results from an Arg 94 to Ser mutation in the second epidermal growth factor (EGF)-like module of factor IX. Recombinant factor IXR94S was activated by factor XIa/calcium with an approximately 50-fold reduced rate and by factor VIIa/tissue factor/phospholipid/calcium with an approximately 20-fold reduced rate compared with wild-type factor IX. The apparent molecular mass of the light chain of factor IXaR94S was approximately 6 kD higher than that of plasma or wild-type factor IX, which was not corrected by N-glycosidase F digestion. This result indicated the presence of additional O-linked carbohydrate in the mutant light chain, probably at new Ser 94. The initial rate of activation of factor X by factor IXaR94S in the presence of polylysine was 7% +/- 1% of the initial rate of activation of factor X by plasma factor IXa, and the kc/Km for activation of factor X by factor IXaR94S/factor VIIIa/phospholipid/calcium was 4% +/- 1% of the kc/Km for activation of factor X by plasma factor IXa/factor VIIIa/phospholipid/calcium. The reduced efficiency of activation of factor X by factor IXaR94S in the tenase enzyme complex was due to a 58-fold +/- 12-fold decrease in kcat with little effect on Km. In conclusion, the R94S mutation had introduced an O-linked carbohydrate, which markedly impaired both activation by factor XIa and turnover of factor X in the tenase enzyme complex.
authors.affiliation: Department of Haematology, Westmead Hospital and University of Sydney, Sydney, New South Wales, Australia. markh@westmed.wh.su.edu.au
authors.forename: M S
authors.initials: MS
authors.lastname: Hertzberg
authors.forename: S L
authors.initials: SL
authors.lastname: Facey
authors.forename: P J
authors.initials: PJ
authors.lastname: Hogg
mesh_terms: D019943:Amino Acid Substitution; D001120:Arginine / Q000235:genetics; D001777:Blood Coagulation / Q000235:genetics; D002241:Carbohydrates; D002384:Catalysis; D004815:Epidermal Growth Factor / Q000235:genetics; D005164:Factor IX / Q000235:genetics* / Q000378:metabolism*; D015942:Factor VIIa / Q000378:metabolism*; D015945:Factor XIa / Q000378:metabolism*; D006801:Humans; D017354:Point Mutation; D012694:Serine / Q000235:genetics; D013925:Thromboplastin / Q000378:metabolism*
chemical_list: D002241:Carbohydrates; D012694:Serine; D004815:Epidermal Growth Factor; D005164:Factor IX; D013925:Thromboplastin; D001120:Arginine; D015942:Factor VIIa; D015945:Factor XIa