A specific disulfide bond associated with the activity of human urokinase. Its topological identification and reductive cleavage followed by kinetic changes in enzymatic reaction and affinity labeling.
A single SH group in the B chain (33 kDa), generated by the specific reduction of the single interchain SS bond of human urinary urokinase, was alkylated (UK X B) with iodoacetamide to prevent a spontaneous SH-SS interchange. An SS bond in UK X B was exclusively alkylated with iodoacetamide (R X CAM-UK X B) after reduction with dithiothreitol in 0.3 M guanidine X HCl in the presence of the competitive inhibitor N alpha-benzoyl-L- argininamide with concomitant loss of 65-68% of the esterolytic activity towards N-acetyl-glycyl-L-lysine methyl ester. This specific SS bond was located at Cys194 - Cys222 whose SS loop contained the active-site Ser198 , as determined by amino acid analyses and identification of the N and C termini of the tryptic digest. Transformation of UK X B into R X CAM-UK X B induced no shift of the optimal pH in the bell-shaped pH/activity profile; pH values for 50% activity were similar (pH 9.7) for 10-min alkalization of the enzyme but different between UK X B (pH 9.4) and R X CAM-UK X B (pH 8.8) for 18-h alkalization. An unaltered Km value and a decline by 64% in kcat in the esterolytic activity indicate that the pretransition Michaelis complex is formed without degeneration of the primary substrate-binding site, but the catalytic pathway thereafter has deteriorated. In affinity labeling with dansyl chloride or N alpha-tosyl-L-lysine chloromethylketone, which interrupted the catalysis at the latest at a stage involving the abortive acyl intermediate, the second-order rate constant for UK X B was lowered to 28% or 35% for R X CAM-UK X B, respectively, but the labeling yields were similar. The results indicate that indispensable structural elements, such as the catalytic triad and oxyanion hole, are maintained but a local conformation, which is necessary for efficient transition to the acyl intermediate and/or for resistance against alkaline inactivation, is destabilized with Cys194 - Cys222 scission.
D000345:Affinity Labels; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D055598:Chemical Phenomena; D002621:Chemistry; D002855:Chromatography, Thin Layer / Q000379:methods; D004220:Disulfides / Q000032:analysis*; D004590:Electrophoresis, Paper; D004950:Esterases / Q000652:urine; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010084:Oxidation-Reduction; D013379:Substrate Specificity; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000652:urine
D000345:Affinity Labels; D000596:Amino Acids; D004220:Disulfides; D004950:Esterases; D014568:Urokinase-Type Plasminogen Activator