Thrombomodulin allosterically modulates the activity of the anticoagulant thrombin.
Exosite 1 of thrombin consists of a cluster of basic residues (Arg-35, Lys-36, Arg-67, Lys-70, Arg-73, Arg-75, and Arg-77 in chymotrypsinogen numbering) that play key roles in the function of thrombin. Structural data suggest that the side chain of Arg-35 projects toward the active site pocket of thrombin, but all other residues are poised to interact with thrombomodulin (TM). To study the role of these residues in TM-mediated protein C (PC) activation by thrombin, a charge reversal mutagenesis approach was used to replace these residues with a Glu in separate constructs. The catalytic properties of the mutants toward PC were analyzed in both the absence and presence of TM and Ca2+. It was discovered that, with the exception of the Arg-67 and Lys-70 mutants, all other mutants activated PC with similar maximum rate constants in the presence of a saturating concentration of TM and Ca2+, although their affinity for interaction with TM was markedly impaired. The catalytic properties of the Arg-35 mutant were changed so that PC activation by the mutant no longer required Ca2+ in the presence of TM, but, instead, it was accelerated by EDTA. Moreover, the activity of this mutant toward PC was improved approximately 25-fold independent of TM. These results suggest that Arg-35 is responsible for the Ca2+ dependence of PC activation by the thrombin-TM complex and that a function for TM in the activation complex is the allosteric alleviation of the inhibitory interaction of Arg-35 with the substrate.
Rezaie
Alireza R
AR
Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, MO 63104, USA. rezaiear@slu.edu
Yang
Likui
L
D000494:Allosteric Regulation; D000818:Animals; D000991:Antithrombins / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D006224:Cricetinae; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011486:Protein C / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013917:Thrombin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018180:Thrombomodulin / Q000378:metabolism*
D000991:Antithrombins; D011486:Protein C; D011994:Recombinant Proteins; D018180:Thrombomodulin; D013917:Thrombin