Previously, we characterized murine mast cell procarboxypeptidase A (MC-proCPA) as an inactive zymogen. To investigate the mechanisms for this lack of enzymatic activity and the processing of the zymogen to the active form, we now have performed molecular modeling of the tertiary structure of murine MC-proCPA based on the x-ray crystallographic structures of porcine pancreatic procarboxypeptidases A and B. Our model predicts that MC-proCPA retains a high degree of structural similarity to its pancreatic homologues. The globular propeptide physically blocks access to the fully formed active site of the catalytic domain and contains a salt bridge to the substrate-binding region that precludes docking of even small substrates. Based on consideration of the predicted tertiary structure and charge field characteristics of the model, the activation site (between GluA94 and Ile1) appears to be highly exposed even after MC-proCPA binds to secretory granule proteoglycans. Based on the steady-state levels of MC-proCPA versus MC-CPA, cycloheximide inhibition of protein synthesis, and brefeldin A blockage of protein sorting, we show that MC-proCPA is processed rapidly in murine mast cell line KiSV-MC14 with a half-life of 26 +/- 5 min (mean +/- S.D., n = 3), and the processing occurs within the secretory granules. The enzyme responsible for this processing may be a thiol protease since treatment of the KiSV-MC14 with 200 microM E-64d, a selective thiol-protease inhibitor, increases MC-proCPA by 2.7 +/- 0.2-fold (mean +/- S.D., n = 3) within 6 h of application.