Evaluating the roles of thrombin and calcium in the activation of coagulation factor XIII using H/D exchange and MALDI-TOF MS.
Factor XIII catalyzes the formation of isopeptide bonds between noncovalently associated fibrin monomers in the final stages of the blood coagulation cascade. This results in a rigid, covalently linked network that is much more resistant to proteolytic degradation. Calcium ion is critical to this process, and its continued presence after activation aids in maintenance of Factor XIII activity. Hydrogen/deuterium exchange experiments were conducted on recombinant Factor XIII a(2) using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The method revealed changes in the structure of Factor XIII a(2) localized to different areas of the protein that were related to the manner in which the enzyme was activated and the calcium environment in which it was maintained. A possible substrate recognition region in the catalytic core (220-230) shows an increase in deuteration upon activation. The degree of deuteration varies depending on the calcium environment in which the active enzyme is maintained. A portion of the beta-sandwich domain (98-104) exhibits a decrease in deuteration upon activation by exposure to calcium alone. A third change occurs in the beta-barrel 1 domain of the protein, a portion of which (526-546) shows a decrease in deuteration upon activation by calcium exposure, but almost none at all when the enzyme is activated by thrombin. The pattern of observed changes reveals individual contributions of calcium and thrombin to activating the enzyme toward substrate binding and exposure of the active site.
Turner
Brian T
BT
Department of Chemistry, University of Louisville, Louisville, KY 40292, USA.
Maurer
Muriel C
MC
D000595:Amino Acid Sequence; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D003903:Deuterium / Q000378:metabolism; D004789:Enzyme Activation; D026122:Factor XIIIa / Q000378:metabolism*; D006801:Humans; D006859:Hydrogen / Q000378:metabolism; D006868:Hydrolysis; D036341:Intercellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D010434:Pepsin A / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013917:Thrombin / Q000737:chemistry* / Q000378:metabolism
D036341:Intercellular Signaling Peptides and Proteins; D010446:Peptide Fragments; D010455:Peptides; C061895:factor XIII activation peptide; D006859:Hydrogen; D003903:Deuterium; D026122:Factor XIIIa; D013917:Thrombin; D010434:Pepsin A; D002118:Calcium