Identification of internal autoproteolytic cleavage sites within the prosegments of recombinant procathepsin B and procathepsin S - Contribution of aplausible unimolecular autoproteolytic event for the processing of zymogens belonging to the papain family

The steps involved in the maturation of proenzymes belonging to the papain family of cysteine proteases have been difficult to characterize. Intermole cular processing at or near the pro/mature junction, due either to the cata lytic activity of active enzyme or to exogeneous proteases, has been well d ocumented for this family of proenzymes. In addition, kinetic studies are s uggestive of a slow unimolecular mechanism of autoactivation which is indep endent of proenzyme concentration. However, inspection of the recently dete rmined x-ray crystal structures does not support this evidence. This is due primarily to the extensive distances between the catalytic thiolate-imidaz olium ion pair and the putative site of proteolysis near the pro/mature jun ction required to form mature protein. Furthermore, the pro-segments for th is family of precursors have been shown to bind through the substrate bindi ng clefts in a direction opposite to that expected for natural substrates. We report, using cystatin C- and N-terminal sequencing, the identification of autoproteolytic intermediates of processing in vitro for purified recomb inant procathepsin B and procathepsin S. Inspection of the x-ray crystal st ructures reported to date indicates that these reactions occur within a seg ment of the proregion which binds through the substrate binding clefts of t he enzymes, thus suggesting that these reactions are occurring as unimolecu lar processes.