The Pro domain of beta-secretase does not confer strict zymogen-like properties but does assist proper folding of the protease domain

beta -Secretase (BACE) is a membrane-bound aspartyl protease that cleaves t he amyloid precursor protein to generate the N terminus of the amyloid P pe ptide. PACE is expressed as a precursor protein containing Pre, Pro, protea se, transmembrane, and cytosolic domains. A soluble PACE derivative (PrePro BACE460) that is truncated between the protease and transmembrane domains w as produced by baculovirus-mediated expression. ProBACE460 was purified fro m conditioned media of infected insect cells using immobilized concanavalin A and immobilized PACE inhibitor, P10-P4 ' Stat(Val), Furin cleaves ProBAC E460 between the Pro and protease regions to generate mature BACE460. The k (cat)/K-m of ProBACE460 when assayed with a polypeptide substrate is only 2 .3-fold less than that of BACE460, This finding and the similar inhibitory potency of P10-P4 ' Stat(Val) for ProBACE460 and BACE460 suggest that the P ro domain has little effect on the PACE active site. Exposure of ProBACE460 to guanidine denaturation/renaturation results in a 7-fold higher recovery of PACE activity than when BACE460 is similarly treated. The presence of f ree PACE Pro peptide during renaturation of BACE460 but not ProBACE460 incr eases recovery of activity. These findings show that the Pro domain in ProB ACE460 does not suppress activity as in a strict zymogen but does appear to facilitate proper folding of an active protease domain.