Conformational changes in human urokinase induced by a specific reduction of disulfide bond in Cys-194-Cys-222 associated with exhibition of enzymatic activity.
The disulfide bond in Cys-194-Cys-222 of the isolated B chain (UK X B) of human urinary urokinase was selectively carboxamidomethylated (R X CAM-UK X B) after specific reduction with dithiothreitol in the presence of the competitive inhibitor N-alpha-benzoyl-L-argininamide (BzArgNH2) and 0.3 M guanidine with inactivation. There were no differences between UK X B and R X CAM-UK X B in CD spectra of 200-250 nm and infrared spectra in the amide I and II regions, whereas two out of five negative CD bands of 250-310 nm were slightly reduced in R X CAM-UK X B. R X CAM-UK X B was more susceptible than UK X B to guanidine denaturation, H-2H exchange in peptide protons and tyrosine-residue ionization. These results indicate that Cys-194-Cys-222 contributes to conformational stabilization against perturbants, but not to static conformation in the resting state, except for a slight disruption in the amino-acid side-chains. BzArgNH2 and 0.3 M guanidine, corresponding to the threshold point toward denaturing transition, enhanced more markedly all the five CD bands of 250-310 nm in UK X B than in R X CAM-UK X B, but did not alter CD spectra of 200-250 nm. The deletion of either BzArgNH2 or guanidine did not induce the inactivation or the cleavage of Cys-194-Cys-222 upon the reduction of UK X B, nor did it induce a marked CD enhancement in the near-ultraviolet upon the addition to UK X B. The results suggest that the BzArgNH2-UK X B interaction enhances the solvent accessibility to the disulfide bond with aid of critical guanidine-denaturation.
D001120:Arginine / Q000031:analogs & derivatives / Q000494:pharmacology; D002942:Circular Dichroism; D004220:Disulfides / Q000032:analysis*; D019791:Guanidine; D006146:Guanidines; D006801:Humans; D007700:Kinetics; D011487:Protein Conformation; D011489:Protein Denaturation; D013055:Spectrophotometry, Infrared; D013329:Structure-Activity Relationship; D014443:Tyrosine; D014568:Urokinase-Type Plasminogen Activator / Q000032:analysis* / Q000378:metabolism
D004220:Disulfides; D006146:Guanidines; D014443:Tyrosine; D001120:Arginine; C038028:N(alpha)-benzoylarginineamide; D014568:Urokinase-Type Plasminogen Activator; D019791:Guanidine