Contribution of residue 192 in factor Xa to enzyme specificity and function.
Mutation of residue 192 (chymotrypsin numbering) from Glu to Gln in thrombin and activated protein C has been shown to dramatically alter substrate and inhibitor specificity, in large part by allowing these enzymes to accept substrates with acidic residues in the P3 and/or P3' positions. In factor Xa, residue 192 is already a Gln. We now compare the properties of a Q192E mutant of Gla-domainless factor X (GDFX). Kinetic analysis of prothrombin activation indicates similar affinity of factor Va for GDFXa and GDFXa Q192E (Kd(app) = 3.6 and 3.7 microM, respectively). Prothrombin activation rates are similar for both enzymes with factor Va, but are approximately 10-fold slower for the Q192E mutant without factor Va. This defect is in the activation of prethrombin 2 and is corrected by factor Va only in the presence of fragment 2. Without factor Va, fragment 2 has no influence on bovine prethrombin 2 activation by GDFXa, but fragment 2 enhances prethrombin 2 activation by the Q192E mutant at least 10-fold. These results indicate that the fragment 2 domain of prothrombin probably alters the conformation of the prethrombin 2 domain, selectively improving its presentation to GDFXa Q192E. With respect to inhibition, tissue factor pathway inhibitor and bovine pancreatic trypsin inhibitor are > or = 30 times poorer inhibitors of GDFXa Q192E than of GDFXa, but these enzymes are inhibited at comparable rates by antithrombin. These results indicate that Gln-192 in factor Xa is a key determinant of substrate/inhibitor specificity.
Rezaie
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AR
Cardiovascular Biology Research Program, University of Oklahoma Health Sciences Center, Oklahoma City, USA.
Esmon
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CT
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism; D015943:Factor Va / Q000378:metabolism; D005170:Factor X / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015951:Factor Xa / Q000235:genetics / Q000378:metabolism*; D065427:Factor Xa Inhibitors; D005973:Glutamine / Q000235:genetics; D006801:Humans; D008074:Lipoproteins / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D011516:Prothrombin / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013917:Thrombin / Q000378:metabolism
D004792:Enzyme Precursors; D065427:Factor Xa Inhibitors; D008074:Lipoproteins; D011994:Recombinant Proteins; C051928:lipoprotein-associated coagulation inhibitor; D005973:Glutamine; D015943:Factor Va; D011516:Prothrombin; D005170:Factor X; D013917:Thrombin; C026812:meizothrombin; D015951:Factor Xa