title: Factor VII mutant V154G models a zymogen-like form of factor VIIa.
abstract: Proteolytic cleavage of the peptide bond between Arg(152) and Ile(153) converts the procoagulant protein Factor VII (FVII) to an activated two-chain form (FVIIa). The formation of a salt bridge between Ile(153) and Asp(343) drives the conversion of FVIIa from being zymogen-like to the active form. In the present paper, we describe the novel FVII mutant V154G (Val(154)-->Gly mutation; residue 17 in the chymotrypsin numbering system), found in three FVII-deficient patients, which models a zymogen-like form of FVIIa. Recombinant V154G FVIIa, although normally cleaved, shows markedly reduced activity towards peptidyl substrate and undetectable activity towards macromolecular substrates. Susceptibility of Ile(153) to chemical modification, in either the presence or the absence of tissue factor (TF), suggests that the reduced V154G FVIIa activity is caused by impaired salt-bridge formation, thus resulting in a zymogen-like FVIIa form. The TF-mediated protection from chemical modification of V154A indicated that Gly(154) is responsible for this peculiar feature, and suggests that this region, proximal to the heavy chain N-terminus, is directly involved in the conversion of FVII into FVIIa. V154G FVII was exploited to study the FVII-TF interaction, together with three additional FVII variants that were expressed to serve as models for different FVII forms. The comparison of binding affinities of full-length TF after relipidation in L-alpha-phosphatidylcholine for the zymogen FVII (Arg(152)-->Gln, K (d)=1.04+/-0.27 nM), inactive FVIIa (Ser(344)-->Ala, K (d)=0.27+/-0.06 nM) and a zymogen-like FVIIa (V154G, K (d)=1.15+/-0.16 nM) supports the hypothesis that preferential binding of TF to active FVIIa is insufficient to drive the 10(5)-fold enhancement of FVIIa activity. In addition, the inability of V154G FVIIa to accommodate an inhibitor in the active site, indicating an improperly shaped specificity pocket, would explain the low activity of the zymogen-like form of FVIIa, which is predominant in the absence of TF.
mesh_terms: D000409:Alanine / Q000235:genetics; D000590:Amino Acid Chloromethyl Ketones / Q000378:metabolism; D001665:Binding Sites; D004792:Enzyme Precursors / Q000737:chemistry / Q000378:metabolism; D005167:Factor VII / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015942:Factor VIIa / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007532:Isoleucine / Q000378:metabolism; D017354:Point Mutation; D011487:Protein Conformation; D015842:Serine Proteinase Inhibitors / Q000378:metabolism; D013925:Thromboplastin / Q000378:metabolism; D014633:Valine / Q000235:genetics*
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D004792:Enzyme Precursors; D015842:Serine Proteinase Inhibitors; D007532:Isoleucine; D005167:Factor VII; D013925:Thromboplastin; D015942:Factor VIIa; D014633:Valine; C040308:phenylalanyl-prolyl-arginine-chloromethyl ketone; D000409:Alanine