From 10,000 to 1: Selective synthesis and enzymatic evaluation of fluorescence resonance energy transfer peptides as specific substrates for chymopapain.

The synthesis and detailed enzymatic analysis of fluorescence resonance energy transfer (FRET)-based peptides as substrates for chymopapain are reported. The design of these substrates arose from a massively parallel high-throughput microarray screening process using peptide nucleic acid (PNA) encoding technology, allowing the identification of detailed substrate specificities of any protease. Two peptides so identified with chymopapain were observed to be excellent substrates with low micromolar K(m) values and turnover numbers on the order of hundreds per second. Mass spectroscopy studies showed unequivocally the specificity of chymopapain toward Ala, Pro, Val, and Lys for positions P(4) to P(1) while not presenting high specificity for residues in position P(1)'.