RESIDUES UNIQUE TO THE PROHORMONE CONVERTASE PC2 MODULATE ITS AUTOACTIVATION, BINDING TO 7B2 AND ENZYMATIC-ACTIVITY

The prohormone convertase PC2 is one of the major subtilisin/kexin-lik e enzymes responsible for the formation of small bioactive peptides in neural and endocrine cells. This convertase is unique among the membe rs of the subtilisin/kexin-like mammalian serine proteinase family in that it undergoes zymogen processing of its inactive precursor proPC2 late along the secretory pathway and requires the help of a PC2-specif ic binding protein known as 7B2, We hypothesized that some of these un ique properties of PC2 are dictated by the presence of PC2-specific am ino acids, which in the six other known mammalian convertases are othe rwise conserved but distinct. Accordingly, six sites were identified w ithin the catalytic segment of PC2, Herein we report on the site-direc ted mutagenesis of Tyr(194) and of the oxyanion hole Asp(309) and the consequences of such mutations on the cellular expression and enzyme a ctivity of PC2, The data show that the Y194D mutation markedly increas es the ex vivo ability of PC2 to process proopiomelanocortin (POMC) in to beta-endorphin in cells devoid of 7B2, e.g. BSC40 cells. In these c ells, expression of native PC2 does not result in the secretion of mea surable in vitro activity against a pentapeptide fluorogenic substrate . In contrast, secreted Y194D-PC2 exhibited significant enzymatic acti vity, even in the absence of 7B2. Based on co-immunoprecipitations and Western blots, binding assays indicate that Tyr(194) participates in the interaction of PC2 with 7B2, and that the oxyanion hole Asp(309) i s critical for the binding of proPC2 with pro7B2. (C) 1998 Federation of European Biochemical Societies.