Interaction of calcium with native and decarboxylated human factor X. Effect of proteolysis in the autolysis loop on catalytic efficiency and factor Va binding.
Human factor X is a two-chain, 58-kDa, vitamin K-dependent blood coagulation zymogen. The light chain of factor X consists of an NH2-terminal gamma-carboxyglutamic acid (Gla) domain, followed by a few helical hydrophobic residues and the two epidermal growth factor-like domains, whereas the heavy chain contains the serine protease domain. In this study, native factor X was found to contain three classes of Ca2+-binding sites: two high affinity (Kd 100 +/- 30 microM), four intermediate affinity (Kd 450 +/- 70 microM), and five to six low affinity (Kd 2 +/- 0.2 mM). Decarboxylated factor X in which the Gla residues were converted to Glu retained the two high affinity sites (Kd 140 +/- 20 microM). In contrast, factor X lacking the Gla domain as well as a part of the helical hydrophobic residues (des-44-X) retained only one high affinity Ca2+-binding site (Kd 130 +/- 20 microM). Moreover, a synthetic peptide composed of residues 238-277 (58-97 in chymotrypsinogen numbering) from the protease domain of factor X bound one Ca2+ with high affinity (Kd 150 +/- 20 microM). From competitive inhibition assays for binding of active site-blocked factor Xa to factor Va in the prothrombinase complex, the Kd for peptide-Va interaction was calculated to be approximately 10 microM as compared with 30 pM for factor Xa and approximately 1.5 microM for decarboxylated factor Xa. A peptide containing residues 238-262(58-82) bound Ca2+ with reduced affinity (Kd approximately 600 microM) and did not inhibit Xa:Va interaction. In contrast, a peptide containing residues 253-277(73-97) inhibited Xa:Va interaction (Kd approximately 10 microM) but did not bind Ca2+. In additional studies, Ca2+ increased the amidolytic activity of native and des-44-Xa toward a tetrapeptide substrate (benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide) by approximately 1.6-fold. The half-maximal increase was observed at approximately 150 microM Ca2+ and the effect was primarily on the kcat. Ca2+ also significantly protected cleavage at Arg-332-Gln-333(150-151) in the protease domain autolysis loop. Des-44-Xa in which the autolysis loop was cleaved possessed </=5% of the amidolytic activity of the noncleaved form; however, the S1 binding site was not affected, as determined by the p-aminobenzamidine binding. Additionally, autolysis loop-cleaved, active site-blocked native factor Xa was calculated to have approximately 10-fold reduced affinity for factor Va as compared with that of the noncleaved form.[1]References
- Interaction of calcium with native and decarboxylated human factor X. Effect of proteolysis in the autolysis loop on catalytic efficiency and factor Va binding. Sabharwal, A.K., Padmanabhan, K., Tulinsky, A., Mathur, A., Gorka, J., Bajaj, S.P. J. Biol. Chem. (1997) [Pubmed]
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