DIFFERENCES IN PH OPTIMA AND CALCIUM REQUIREMENTS FOR MATURATION OF THE PROHORMONE CONVERTASES PC2 AND PC3 INDICATES DIFFERENT INTRACELLULAR LOCATIONS FOR THESE EVENTS

PC2 and PC3, which is also known as PC1, are subtilisin-like proteases that are involved in the intracellular processing of prohormones and proneuropeptides. Both enzymes are synthesized as propolypeptides that undergo proteolytic maturation within the secretory pathway. An in vi tro translation/translocation system from Xenopus egg extracts was use d to investigate mechanisms in the maturation of pro-PC3 and pro-PC2. Pro-PC3 underwent rapid (t(1/2) < 10 min) processing of the 88-kDa pro polypeptide at the sequence RSKR(83) to generate the 80-kDa active for m of the enzyme. This processing was blocked when the active site aspa rtate was changed to asparagine, suggesting that an autocatalytic mech anism was involved. In this system, processing of pro-PCS was optimal between pH 7.0 and 8.0 and was not dependent on additional calcium. Th ese results are consistent with pro-PCS maturation occurring at an ear ly stage in the secretory pathway, possibly within the endoplasmic ret iculum, where the pH would be close to neutral and the calcium concent ration less than that observed in later compartments. Processing of pr o-PC2 in the Xenopus egg extract was much slower than that of pro-PC3 (t(1/2) = 8 h). It exhibited a pH optimum of 5.5-6.0 and was dependent on calcium (K-0.5 = 2-4 mM), The enzymatic properties of pro-PCB proc essing were similar to that of the mature enzyme. Further studies usin g mutant pro PCB constructs suggested that cleavage of pro-PC2 was cat alyzed by the mature 68-kDa PC2 molecule. The results were consistent with pro-PC2 maturation occurring within a late compartment of the sec retory pathway that contains a high calcium concentration and low pH.