Intrinsic pathway activation of factor X and its activation peptide-deficient derivative, factor Xdes-143-191.
The role of the activation peptide in determining the substrate specificity of intrinsic pathway factor X (fX) activation was studied by using a novel derivative of fX in which 49 residues were removed enzymatically from the NH2 terminus of the 52-residue activation peptide by an enzyme from the venom of the snake Agkistrodon rhodostoma. The modified protein, designated fXdes-143-191, is inactive but is activated to alpha-fXa by either the intrinsic fX activation complex (intrinsic fXase) composed of factor IXa beta, thrombin-activated factor VIII (fVIIIaIIa), and phospholipid vesicles or by the fX coagulant protein from Russell's viper venom (RVV-XCP). Both the Km and kcat for the activation of fX by RVV-XCP were greater than for fXdes-143-191, resulting in less than a 2-fold difference in the catalytic efficiency (kcat/Km) suggestive of nonproductive binding of fXdes-143-191 to RVV-XCP. The activation of each substrate by intrinsic fXase revealed that the kcat was 100-fold greater for fX than fXdes-143-191 (16 and 0.16 s-1, respectively), although there was no detectable difference in Km (60 and 80 nM, respectively). Activations by fIXa beta/phospholipid in the absence of fVIIIaIIa also revealed a difference in kcat but not Km, but the difference in kcat was smaller (kcat of 0.007 and 0.002 s-1 and Km of 220 and 170 nM for fX and fXdes-143-191, respectively). Analysis of product versus time curves demonstrated that fVIIIaIIa promotes formation of the actyl-enzyme intermediate during fX activation. We conclude that the activation peptide plays a critical role during acyl-enzyme formation that is most pronounced in the presence of fVIIIaIIa. The absence of Km differences suggests that residues NH2-terminal to P3 do not contribute to the initial formation of the enzyme-substrate complex.
Duffy
E J
EJ
Department of Medicine, Emory University, Atlanta, Georgia 30322.
Lollar
P
P
D000595:Amino Acid Sequence; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000494:pharmacology; D005169:Factor VIII / Q000378:metabolism; D005170:Factor X / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D013379:Substrate Specificity; D013552:Swine
D010446:Peptide Fragments; D005169:Factor VIII; D005170:Factor X; D010450:Endopeptidases; D008666:Metalloendopeptidases; C033858:russellysin