Astacin, a zinc-endopeptidase from the crayfish Astacus astacus, hydrolyzes the pentaalanine substrate Suc-AAAAA-pNA at two positions. The first, highly specific cleavage yields Suc-Ala-Ala and Ala-Ala-Ala-pNA (L-1090); the second, much less specific cleavage releases p-nitroaniline from the generated chromogenic substrate.