Thermodynamic linkage between the S1 site, the Na+ site, and the Ca2+ site in the protease domain of human coagulation factor xa. Studies on catalytic efficiency and inhibitor binding.
The serine protease domain of factor Xa (FXa) contains a sodium as well as a calcium-binding site. Here, we investigated the functional significance of these two cation-binding sites and their thermodynamic links to the S1 site. Kinetic data reveal that Na(+) binds to the substrate bound FXa with K(d) approximately 39 mm in the absence and approximately 9.5 mm in the presence of Ca(2+). Sodium-bound FXa (sodium-Xa) has approximately 18-fold increased catalytic efficiency ( approximately 4.5-fold decrease in K(m) and approximately 4-fold increase in k(cat)) in hydrolyzing S-2222 (benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide), and Ca(2+) further increases this k(cat) approximately 1.4-fold. Ca(2+) binds to the protease domain of substrate bound FXa with K(d) approximately 705 microm in the absence and approximately 175 microm in the presence of Na(+). Ca(2+) binding to the protease domain of FXa (Xa-calcium) has no effect on the K(m) but increases the k(cat) approximately 4-fold in hydrolyzing S-2222, and Na(+) further increases this k(cat) approximately 1.4-fold. In agreement with the K(m) data, sodium-Xa has approximately 5-fold increased affinity in its interaction with p-aminobenzamidine (S1 site probe) and approximately 4-fold increased rate in binding to the two-domain tissue factor pathway inhibitor; Ca(2+) (+/-Na(+)) has no effect on these interactions. Antithrombin binds to Xa-calcium with a approximately 4-fold faster rate, to sodium-Xa with a approximately 24-fold faster rate and to sodium-Xa-calcium with a approximately 28-fold faster rate. Thus, Ca(2+) and Na(+) together increase the catalytic efficiency of FXa approximately 28-fold. Na(+) enhances Ca(2+) binding, and Ca(2+) enhances Na(+) binding. Further, Na(+) enhances S1 site occupancy, and S1 site occupancy enhances Na(+) binding. Therefore, Na(+) site is thermodynamically linked to the S1 site as well as to the protease domain Ca(2+) site, whereas Ca(2+) site is only linked to the Na(+) site. The significance of these findings is that during physiologic coagulation, most of the FXa formed will exist as sodium-Xa-calcium, which has maximum biologic activity.
Underwood
M C
MC
Department of Biochemistry and Molecular Biology and the Departments of Medicine and Pathology, St. Louis University School of Medicine, St. Louis, Missouri 63104, USA.
Zhong
D
D
Mathur
A
A
Heyduk
T
T
Bajaj
S P
SP
D010129:4-Aminobenzoic Acid / Q000378:metabolism; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002384:Catalysis; D004357:Drug Synergism; D010450:Endopeptidases / Q000378:metabolism*; D015951:Factor Xa / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008074:Lipoproteins / Q000378:metabolism; D008958:Models, Molecular; D009842:Oligopeptides / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D012964:Sodium / Q000378:metabolism*; D013816:Thermodynamics
D008074:Lipoproteins; D009842:Oligopeptides; C051928:lipoprotein-associated coagulation inhibitor; C015608:benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide; D012964:Sodium; D010450:Endopeptidases; D015951:Factor Xa; D002118:Calcium; D010129:4-Aminobenzoic Acid