The effect of interaction between human urokinase and its competitive inhibitor, N alpha-benzoyl-L-arginine amide, on reduction of a specific SS bond related to enzymatic activity.
The 55- (H-UK) and 36-kDa forms (L-UK) of human urinary urokinase lost most of esterase activity toward acetyl-glycyl-L-lysine methyl ester upon reductive cleavage of 3 SS bonds with dithiothreitol in the presence of the competitive inhibitor, N alpha-benzoyl-L-arginine amide (BAA), bound to polyacrylyl azide with C16N3-arm (PAA) at 0.3 M guanidine, a threshold point of the native state where a protein-denaturating transition began. One of the 3 SS bonds was protected from reduction, with an unaltered activity, under the similar conditions except for replacement of BAA-PAA conjugate by glycine-PAA conjugate. This "specific" SS bond was reduced and, after the other SH groups produced were blocked with iodoacetamide (IAM), selectively reoxidized, which resulted in complete reactivation. The intact B-chain isolated from H-UK was completely inactivated when its specific SS bond was reduced and selectively alkylated with IAM after the other SH groups were reversibly blocked with 5, 5'-dithiobis (2-nitrobenzoic acid), which was finally removed. The results indicate that a single specific SS bond is essential for retaining a conformation necessary to activity exhibition.
D001120:Arginine / Q000031:analogs & derivatives* / Q000494:pharmacology; D001665:Binding Sites / Q000187:drug effects; D001667:Binding, Competitive; D004220:Disulfides / Q000378:metabolism; D006801:Humans; D010084:Oxidation-Reduction; D011480:Protease Inhibitors; D011489:Protein Denaturation; D012997:Solvents; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors*
D004220:Disulfides; D011480:Protease Inhibitors; D012997:Solvents; D001120:Arginine; C038028:N(alpha)-benzoylarginineamide; D014568:Urokinase-Type Plasminogen Activator