Probing the molecular basis of factor Xa specificity by mutagenesis of the serpin, antithrombin.
The molecular basis of the substrate and inhibitor specificity of factor Xa, the serine proteinase of the prothrombinase complex, was investigated by constructing two mutants of human antithrombin (HAT) in which the reactive site loop of the serpin from the P4-P4' site was replaced with the corresponding residues of the two factor Xa cleavage sites in prothrombin (HAT/Proth-1 and HAT/Proth-2). These mutants together with prethrombin-2, the smallest zymogen form of thrombin containing only the second factor Xa cleavage site, were expressed in mammalian cells, purified to homogeneity and characterized in kinetic reactions with factor Xa in both the absence and presence of cofactors; factor Va, high affinity heparin and pentasaccharide fragment of heparin. HAT/Proth-1 inactivated factor Xa approximately 3-4-fold better than HAT/Proth-2 in either the absence or presence of heparin cofactors. In the absence of a cofactor, factor Xa reacted with the HAT/Proth-2 and prethrombin-2 with similar second-order rate constants (approximately 2-3x10(2) M(-1)s(-1)). Pentasaccharide catalyzed the inactivation rate of factor Xa by the HAT mutants 300-500-fold. A similar 10(4)-10(5)-fold enhancement in the reactivity of factor Xa with prethrombin-2 and the HAT mutants was observed in the presence of the cofactors Va and heparin, respectively. Factor Va did not influence the reactivity of factor Xa with either one of the HAT mutants. These results suggest that (1) in the absence of a cofactor, the P4-P4' residues of HAT and prethrombin-2 primarily determine the specificity reactions with factor Xa, (2) factor Va binding to factor Xa is not associated with allosteric changes in the catalytic pocket of enzyme that would involve interactions with the P4-P4' binding sites, and (3) similar to allosteric activation of HAT by heparin, a role for factor Va in the prothrombinase complex may involve rearrangement of the residues surrounding the scissile bond of the substrate to facilitate its optimal docking into the catalytic pocket of factor Xa.
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Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, 1402 South Grand Boulevard, St. Louis, MO 63104, USA. rezaiear@slu.edu
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D000991:Antithrombins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D004792:Enzyme Precursors / Q000378:metabolism; D005165:Factor V / Q000378:metabolism; D015943:Factor Va / Q000494:pharmacology; D015951:Factor Xa / Q000378:metabolism*; D065427:Factor Xa Inhibitors; D005822:Genetic Vectors; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D009154:Mutation; D010446:Peptide Fragments / Q000494:pharmacology; D011516:Prothrombin / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification; D016415:Sequence Alignment
D000991:Antithrombins; D004792:Enzyme Precursors; D065427:Factor Xa Inhibitors; D010446:Peptide Fragments; D011994:Recombinant Proteins; C022475:prothrombinase complex; D015943:Factor Va; D005165:Factor V; D011516:Prothrombin; D006493:Heparin; C012007:prethrombins; D015951:Factor Xa