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.
Sabharwal
A K
AK
Department of Medicine, Saint Louis University School of Medicine, St. Louis, Missouri 63104, USA.
Padmanabhan
K
K
Tulinsky
A
A
Mathur
A
A
Gorka
J
J
Bajaj
S P
SP
D015055:1-Carboxyglutamic Acid / Q000378:metabolism; D001329:Autolysis; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002384:Catalysis; D015943:Factor Va / Q000378:metabolism*; D005170:Factor X / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D011485:Protein Binding; D011516:Prothrombin / Q000378:metabolism; D013324:Strontium / Q000378:metabolism; D013329:Structure-Activity Relationship
D015055:1-Carboxyglutamic Acid; D015943:Factor Va; D011516:Prothrombin; D005170:Factor X; D002118:Calcium; D013324:Strontium