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 (Cia) 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 (K-d 100 +/- 30 mu M), four intermediate affinity (K-d 450 +/- 70 mu M), and five to six low affinity (K-d 2 +/- 0.2 mm). Decarboxylated factor X in which the Gla residues were converted to Glu retained the two high affinity sites (K-d 140 +/- 20 mu M). 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 (K-d 130 +/- 20 mu M). 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 (K-d 150 +/- 20 mu M). From competitive inhibition assays for binding of active site-blocked factor Xa to factor Va in the prothrombinase complex, the K-d for peptide-Va interaction was calculated to be similar to 10 mu M as compared with 30 phn for factor Xa and similar to 1.5 mu M for decarboxylated factor Xa, A peptide containing residues 238-262 (58-82) bound Ca2+ with reduced affinity (K-d similar to 600 mu M) and did not inhibit Xa:Va interaction, In contrast, a peptide containing residues 253-277(73-97) inhibited Xa:Va interaction (K-d similar to 10 mu M) 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 similar to 150 mu M Ca2+ and the effect was primarily on the k(cat). 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 less than or equal to 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 similar to 10-fold reduced affinity for factor Va as compared with that of the noncleaved form.