Na+ site in blood coagulation factor IXa: effect on catalysis and factor VIIIa binding.
During blood coagulation, factor IXa (FIXa) activates factor X (FX) requiring Ca2+, phospholipid, and factor VIIIa (FVIIIa). The serine protease domain of FIXa contains a Ca2+ site and is predicted to contain a Na+ site. Comparative homology analysis revealed that Na+ in FIXa coordinates to the carbonyl groups of residues 184A, 185, 221A, and 224 (chymotrypsin numbering). Kinetic data obtained at several concentrations of Na+ and Ca2+ with increasing concentrations of a synthetic substrate (CH3-SO2-d-Leu-Gly-Arg-p-nitroanilide) were fit globally, assuming rapid equilibrium conditions. Occupancy by Na+ increased the affinity of FIXa for the synthetic substrate, whereas occupancy by Ca2+ decreased this affinity but increased k(cat) dramatically. Thus, Na+-FIXa-Ca2+ is catalytically more active than free FIXa. FIXa(Y225P), a Na+ site mutant, was severely impaired in Na+ potentiation of its catalytic activity and in binding to p-aminobenzamidine (S1 site probe) validating that substrate binding in FIXa is linked positively to Na+ binding. Moreover, the rate of carbamylation of NH2 of Val16, which forms a salt-bridge with Asp194 in serine proteases, was faster for FIXa(Y225P) and addition of Ca2+ overcame this impairment only partially. Further studies were aimed at delineating the role of the FIXa Na+ site in macromolecular catalysis. In the presence of Ca2+ and phospholipid, with or without saturating FVIIIa, FIXa(Y225P) activated FX with similar K(m) but threefold reduced k(cat). Further, interaction of FVIIIa:FIXa(Y225P) was impaired fourfold. Our previous data revealed that Ca2+ binding to the protease domain increases the affinity of FIXa for FVIIIa approximately 15-fold. The present data indicate that occupancy of the Na+ site further increases the affinity of FIXa for FVIIIa fourfold and k(cat) threefold. Thus, in the presence of Ca2+, phospholipid, and FVIIIa, binding of Na+ to FIXa increases its biologic activity by approximately 12-fold, implicating its role in physiologic coagulation.
Schmidt
Amy E
AE
UCLA/Orthopaedic Hospital, Department of Orthopaedic Surgery and Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA.
Stewart
Jonathan E
JE
Mathur
Akash
A
Krishnaswamy
Sriram
S
Bajaj
S Paul
SP
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002384:Catalysis / Q000187:drug effects; D002414:Cations, Monovalent / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D015949:Factor IXa / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015944:Factor VIIIa / Q000737:chemistry* / Q000378:metabolism*; D005170:Factor X / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding / Q000187:drug effects; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D012964:Sodium / Q000378:metabolism* / Q000494:pharmacology*; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism; D014633:Valine / Q000235:genetics / Q000378:metabolism
D002414:Cations, Monovalent; D014443:Tyrosine; D015944:Factor VIIIa; D005170:Factor X; D012964:Sodium; D015949:Factor IXa; D014633:Valine; D002118:Calcium