Herein we investigated the processing of beta-secretase (BACE), implicated in Alzheimer's disease through processing of beta-amyloidprecursor protein (betaAPP), into smaller metabolites. Four products of similar to34, similar to12, similar to8, and similar to5 kDa were identified, none of which were generated autocatalytically. The similar to34 and similar to12 kDa forms are held together by disulfide bridges. The similar to34 kDa form results from two cleavages: an N-terminal processing at RLPR(45)down arrow by furin/PC5, and a C-terminal cleavage at SQDD(379)down arrow by an unknown enzyme that also releases the C-terminal similar to12 kDa product. Microsequencing of the similar to8 and similar to5 kDa fragments showed that they are the result of processing at VVFD(407)down arrow and DMED(442)down arrow, respectively. Mutagenesis of the identified cleavage sites revealed that the mutants D379A, D379L or D379E block the degradation of BACE into the similar to12 kDa product, confirming the importance of ASP379. Notably, the D379E mutant results in higher betaAPP derived C99 levels. In contrast, D442A or D442E did not affect the production of the similar to8 or similar to5 kDa products. The levels of the similar to8 and similar to5 kDa products are significantly lower in the mutant D407A but less so D407E, likely due to the low efficacy of ER exit of the D407A mutant. Indeed, while co-expression of betaAPP with BACE results in enhanced production of Abeta(11-40), the D407A mutant produces mostly Abeta(40). (C) 2004 Elsevier Inc. All rights reserved.