The proteolytic maturation of prohormone convertase 2 (PC2) is a pH-drivenprocess

Recombinant proPC2 purified from the medium of CHO cells overexpressing bot h the prohormone convertase (PC) precursor proPC2 and the 21-kDa amino term inal portion of the neuroendocrine protein 7B2 can spontaneously convert to an active species. In the present report, we have characterized the proPC2 zymogen conversion process. Sequencing of the mature 66 kDa enzyme reveale d a single site of cleavage at the paired basic site amino terminal to the GYRDI sequence. In contrast to mature PC2 activity, proPC2 conversion was i nhibited neither by the eukaryotic subtilisin inhibitor pCMS nor by the spe cific PC2 inhibitor, 7B2 CT peptide, suggesting significant differences bet ween the proPC2 conversion reaction and the hydrolysis of synthetic substra tes by mature PC2, In support of this idea, proPC2 conversion was not calci um dependent and was unaffected by 5 mM EDTA. The rate of conversion of pro PC2 remained similar with a 10-fold difference in zymogen concentration, im plicating an intramolecular rather than intermolecular mechanism of activat ion Interestingly, the rate of proPC2 conversion was extremely pH dependent , occurring most extensively between pHs 4.0 and 4.9. Taken together, our r esults suggest that cellular proPC2 maturation occurs via an autocatalytic, intramolecular process controlled not by 7B2 inhibition nor by calcium lev els, but by the decreasing pH gradient along the secretory pathway. (C) 199 9 Academic Press.