Designing an enzyme requires, among a number of parameters, the appropriate positioning of catalytic machinery within a substrate-binding cleft. Using the structures of cyclophilin-peptide complexes(1-4), we have engineered a new catalytic activity into an Escherichia coli cyclophilin by mutating three amino acids, close to the peptide binding deft, to form a catalytic triad similar to that found in serine proteases. In conjunction with cyclophilin＇s specificity for proline-bearing peptides, this creates a unique endopeptidase, cyproase 1, which cleaves peptides on the amino-side of proline residues. When acting on an Ala-Pro dipeptide, cyproase 1 has an efficiency (k(cat)/K-m) of 0.7 x 10(4) M-1 s(-1) and enhances the rate of reaction (k(cat)/k(uncat)) 8 x 10(8)-fold. This activity depends upon a deprotonated histidine and is inhibited by nucleophile-specific reagents, as occurs in natural serine proteases. Cyproase 1 can hydrolyse a protein substrate with a proline-specific endoprotease activity.