The structure of tripeptidylpeptidase II (TPPII) has shown that it belongs to the group of exopeptidases which use a double-Glu motif to convey aminopeptidase activity. TPPII has been implicated in vital biological processes. At least one of these, antigen processing, requires the involvement of its endopeptidase activity. In order to understand the extent and molecular basis of this unusual functional promiscuity we have performed a systematic kinetic analysis of wild type Drosophila melanogaster TPPII and five point mutants of the double-Glu-motif (E312/E343) involving natural substrates. Unlike the known double-Glu motives of other exopeptidases, the double-Glu motif of TPPII is distinctly asymmetrical: E312 is the crucial determinant of the aminotripeptidolytic ruler mechanism. It both blocks the active-site cleft at substrate position P4 and forms a salt bridge with the N-terminus of the substrate. In contrast, E343 forms a much weaker salt bridge than E312 and it does not have a blocking role. An endopeptidase substrate can bind at relatively high affinity if the length of the substrate permits binding to several S' sites. However, the lacking alignment of the substrate by the double-Glu motif causes the endopeptidolytic K(cat)/K(M) of TPPII to be very low. (C) 2011 Elsevier Inc. All rights reserved.