Difference in enzymatic properties between "staphylothrombin" and free alpha-thrombin.
Bacterial protein, staphylocoagulase, binds stoichiometrically to human prothrombin resulting in a coagulant complex, staphylothrombin. The enzymatic properties of staphylothrombin differ from those of alpha-thrombin in their substrate specificities toward natural and synthetic substrates, in addition to their interaction with protease inhibitors. In order to obtain information about the region of staphylocoagulase that interacts with human prothrombin, staphylocoagulase was cleaved by alpha-chymotrypsin. This limited alpha-chymotryptic cleavage of staphylocoagulase yielded three large fragments, fragments of 43, 30, and 20 kDa. The 43-kDa fragment exhibited a high affinity for human prothrombin (Kd = 1.7 nM), which is comparable to the affinity observed using intact staphylocoagulase (Kd = 0.46 nM). A complex of the 43-kDa fragment and prothrombin possessed both clotting and amidase activities essentially identical to those observed in a complex of intact staphylocoagulase and prothrombin. The 30-kDa fragment exhibited weaker affinity for prothrombin (Kd = 120 nM). While a complex of this fragment and prothrombin did not exhibit clotting activity, it nonetheless possessed a weak amidase activity. The 20-kDa fragment was found only to bind to prothrombin. The NH2-terminal sequence analyses of these fragments revealed that the 43-kDa fragment constitutes the NH2-terminal portion of staphylocoagulase, and contains the 30-kDa and 20-kDa fragments. It is therefore concluded that the functional region of staphylocoagulase for binding and activation of prothrombin is localized in the NH2-terminal region of the intact protein. The 43-kDa fragment contains 324 amino acids with a molecular weight of 38,098. The 43-kDa fragment has an unusual amino acid composition based on the sequence, in which the sum of Asp (28 residues), Asn (22), Glu (35), Gln (9), and Lys (52) residues accounts for more than 45% of the total residues. A comparison of the amino acid sequence of the 43-kDa fragment with that of streptokinase did not reveal any obvious sequence homology. There was also no sequence homology with those of trypsin, alpha-chymotrypsin, and elastase.
D001665:Binding Sites; D001777:Blood Coagulation; D003030:Coagulase / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D011487:Protein Conformation; D011516:Prothrombin / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism*
D003030:Coagulase; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011480:Protease Inhibitors; D011516:Prothrombin; D010447:Peptide Hydrolases; C023073:tosylarginine methyl ester hydrolase; D013917:Thrombin