Substrate Profiling of Cysteine Proteases Using a Combinatorial Peptide Library Identifies Functionally Unique Specificities*□S

CA, family C1) papain, bromelain, and human cathepsins L, V, K, S, F, B, and five proteases of parasitic origin were studied using a com-pletely diversified positional scanning synthetic combinatorial library. A bifunctional coumarin fluorophore was used that facili-tated synthesis of the library and individual peptide substrates. The library has a total of 160,000 tetrapeptide substrate sequences com-pletely randomizing each of the P1, P2, P3, and P4 positions with 20 amino acids. Amicrotiter plate assay format permitted a rapid determination of the specificity profile of each enzyme. Individ-ual peptide substrates were then synthesized and tested for a quantitative determination of the specificity of the human cathe-psins. Despite the conserved three-dimensional structure and similar substrate specificity of the enzymes studied, distinct amino acid preferences that differentiate each enzyme were identified. The specificities of cathepsins K and S partially matc