A loop of coagulation factor VIIa influencing macromolecular substrate specificity.
Coagulation factor VIIa (FVIIa) belongs to a family of proteases being part of the stepwise, self-amplifying blood coagulation cascade. To investigate the impact of the mutation Met(298{156})Lys in FVIIa, we replaced the Gly(283{140})-Met(298{156}) loop with the corresponding loop of factor Xa. The resulting variant exhibited increased intrinsic activity, concurrent with maturation of the active site, a less accessible N-terminus, and, interestingly, an altered macromolecular substrate specificity reflected in an increased ability to cleave factor IX (FIX) and a decreased rate of FX activation compared to that of wild-type FVIIa. In complex with tissue factor, activation of FIX, but not of FX, returned to normal. Deconvolution of the loop graft in order to identify important side chain substitutions resulted in the mutant Val(158{21})Asp/Leu(287{144})Thr/Ala(294{152})Ser/Glu(296{154}) Ile/Met(298{156})Lys-FVIIa with almost the same activity and specificity profile. We conclude that a lysine residue in position 298{156} of FVIIa requires a hydrophilic environment to be fully accommodated. This position appears critical for substrate specificity among the proteases of the blood coagulation cascade due to its prominent position in the macromolecular exosite and possibly via its interaction with the corresponding position in the substrate (i.e. FIX or FX).
Bjelke
Jais R
JR
Protein Structure and Biophysics, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark. jarb@novonordisk.com
Persson
Egon
E
Rasmussen
Hanne B
HB
Kragelund
Birthe B
BB
Olsen
Ole H
OH
D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D001777:Blood Coagulation / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D015942:Factor VIIa / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015951:Factor Xa / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics; D013925:Thromboplastin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D009097:Multienzyme Complexes; D013925:Thromboplastin; D015942:Factor VIIa; D015951:Factor Xa