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.