The reaction of bovine thrombin with N-butyrylimidazole. Two different reactions resulting in the inhibition of catalytic activity.
N-Butyrylimidazole has been found to be a potent inhibitor of purified bovine thrombin. The rate and extent of inhibition of thrombin by N-butyrylimidazole could be reduced by the presence of benzamidine, a competitive inhibitor, or by the ester substrate, p-tosyl-L-arginine methyl ester. Spectral studies of the reaction of N-butyrylimidazole with thrombin demonstrated the modification of approximately 1 mol of tyrosine/mol of enzyme at maximum inhibition. In addition to the reaction with tyrosine, N-butyrylimidazole also appears to react with a residue at the "active site" as judged by a decrease in the number of active sites available in the modified enzyme for titration with p-nitrophenyl-p'-guanidinobenzoate. The time course of ester hydrolysis by butyrylated thrombin showed a distinct lag phase suggesting partial reactivation of the enzyme under assay conditions. Partial reactivation of the modified enzyme also occurred spontaneously upon standing in 0.5 M NaCl but was much faster in presence of imidazole (0.03 M, pH 7.6). It is suggested that, in addition to reaction with tyrosine, there is a reaction of N-butyrylimidazole with either the histidine and/or serine residue at the active site of thrombin resulting in a derivative unstable under esterase assay conditions such as that described for the reaciton of N-acetylimidazole with trypsin (L. L. Houston and K. A. Walsh (1970), Biochemistry 9, 156).
D000818:Animals; D001665:Binding Sites; D002087:Butyrates; D002417:Cattle; D004789:Enzyme Activation / Q000187:drug effects; D007093:Imidazoles / Q000494:pharmacology; D007700:Kinetics; D011485:Protein Binding; D012965:Sodium Chloride / Q000494:pharmacology; D013917:Thrombin / Q000037:antagonists & inhibitors; D013997:Time Factors; D014106:Tosylarginine Methyl Ester; D014443:Tyrosine
D002087:Butyrates; D007093:Imidazoles; D014443:Tyrosine; D012965:Sodium Chloride; D014106:Tosylarginine Methyl Ester; D013917:Thrombin