Cathepsin B and other lysosomal cysteine proteinases are synthesized as inactive zymogens, which are converted to their mature forms by other proteases or by autocatalytic processing. Procathepsin B autoactivation was shown in vitro at pH 4.5 to be a bimolecular process with K s and k cat values of 2.1 þ 0.9 W WM and 0.12 þ 0.02 s 31 , respectively. Autoactivation is substantially accelerated in the presence of active cathepsin B molecules, indicating that mature cathepsin B is the catalytic species in the process. Proenzyme is cleaved without significant conformational changes as judged by circular dichroism, suggesting that propeptide unfolding occurs only after the cleavage. Procathepsin B autoactivation is pH-dependent with a pH optimum at 4.5 and with no processing observed at pH s 6.0. However, in the presence of 0.5 W Wg/ml of dextran sulfate, relatively rapid processing is observed even at pH 6.5 (t 1a2 V V90 min), suggesting that glycosaminoglycans are involved in in vivo processing of lysosomal cysteine proteases.z 1999 Federation of European Biochemical Societies.
Procathepsin B undergoes autocatalytic processing to its active form after exposure to acidic pH. In the present study the effect of pH, proenzyme concentration and dextran sulphate on the processing of recombinant human procathepsin B was investigated in order to determine the mechanism of this process. The processing was followed fluorimetricaly, using Z-Arg-Arg-AMC as a substrate for cathepsin B. We obtained sigmoidal curves, which were concentration dependent, indicating that the processing of cathepsin B is a bimolecular process. The K, and kat values of 4.1pM and 2 . 4~1 0 "~ respectively, were obtained by simultaneus nonlinear regression analysis of 15 data sets. Processing of procathepsin B was also followed by SDS-PAGE. Activation was completed within 30 minutes, in agreement with simulation experiments at high procathepsin B concentration (1 8-8OnM). Changes in tertiary and secondary structures during the processing were followed by circular dichroism measurements of procathepsin B, cathepsin B and mutant inactive procathepsin B(C25S).
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