Deletion of the 60-loop provides new insights into the substrate and inhibitor specificity of thrombin.
Structural data have indicated that the 60-loop of thrombin with 8-9 insertion residues is responsible for the restricted substrate and inhibitor specificity of thrombin. However,previous deletion of 3-4 residues of this loop (des-PPW and des-YPPW) did not widen the specificity of thrombin, but further restricted it. The partial deletion of this loop also dramatically impaired the reactivity of thrombin with antithrombin (AT), protein C and fibrinogen, implicating a role for the productive interaction of the 60-loop with the target macromolecules. To further investigate the role of this loop, a mutant of thrombin was expressed in mammalian cells in which all 8 residues (Tyr-Pro-Pro-Trp-Asp-Lys-Asn-Phe) of the 60-loop were deleted (des-60-loop). In contrast to the partially deleted loop mutants, it was discovered that the des-60-loop mutant cleaved small synthetic substrates, clotted purified fibrinogen, and activated protein C with a near normal catalytic efficiency; however, its activity toward cofactors V and VIII was impaired approximately 2-4-fold. Direct binding and AT inhibition studies in the presence of heparin revealed that the affinity of heparin for interaction with exosite-2 of des-60-loop thrombin was impaired, though the reactivity of the mutant with AT and other plasma serpins was not impaired, but rather improved approximately 2-fold. These results suggest that the 60-loop plays a key role in regulating the specificity of thrombin by shielding the active-site pocket, but its productive interaction with the target molecules may not be as critical as has been speculated in previous reports.
Rezaie
Alireza R
AR
Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104, USA. rezaiear@slu.edu
Yang
Likui
L
D000818:Animals; D001777:Blood Coagulation / Q000187:drug effects; D020134:Catalytic Domain; D002460:Cell Line; D002863:Chromogenic Compounds; D006224:Cricetinae; D004789:Enzyme Activation; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D011486:Protein C / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D017384:Sequence Deletion; D012964:Sodium / Q000378:metabolism; D013379:Substrate Specificity; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D002863:Chromogenic Compounds; D011486:Protein C; D011994:Recombinant Proteins; D012964:Sodium; D013917:Thrombin