Memapsin 2 is the protease known as β-secretase whose action on β-amyloid precursor protein leads to the production of the β-amyloid (Aβ) peptide. Since the accumulation of Aβ in the brain is a key event in the pathogenesis of Alzheimer's disease, memapsin 2 is an important target for the design of inhibitory drugs. Here we describe the residue preference for the subsites of memapsin 2. The relative kcat/KM values of residues in each of the eight subsites were determined by the relative initial cleavage rates of substrate mixtures as quantified by MALDI-TOF mass spectrometry. We found that each subsite can accommodate multiple residues. The S1 subsite is the most stringent, preferring residues in the order of Leu > Phe > Met > Tyr. The preferences of other subsites are the following: S2, Asp > Asn > Met; S3, Ile > Val > Leu; S4, Glu > Gln > Asp; S1‘, Met > Glu > Gln > Ala; S2‘, Val > Ile > Ala; S3‘, Leu > Trp > Ala; S4‘, Asp > Glu > Trp. In general, S subsites are more specific than the S‘ subsites. A pe...