Combinatorial peptide library for probing the selectivity of the s-1 subsite of proteases

A combinatorial tetrapeptide library, Suc-Ala-Phe-Arg-AA(1)-OR, in which R = p-formamidobenzyl ester and AA(1) = 17 of the 20 natural occurring amino acids, has been synthesized chemically and separated by a reverse phase HPL C. The library was used to study the s-1 subsite specificity of various pro teases. The preferred substrate at the s-1 subsite of chymotrypsin is in th e order of Trp > Tyr > Phe > Met > Leu. This agreed with the reported data that the favored substrate at the s-1 subsite for chymotrypsin-catalyzed hy drolysis is an aromatic amino acid residue. The hydrophobic amino acid resi dues at this subsite can be hydrolysized after a longer incubating time. Th is procedure of selective hydrolysis of a peptide library was used to probe the selectivity of s-1 subsites of four proteases isolated from Bacillus s tearothermophilus, subtilisin Carlsberg, subtilisin BPN' and an engineered protease subtilisin 8397. The protease from Bacillus stearothermophilus fav ored the substrate with residue Lys, and Arg at the s-1 subsite as a trypsi n-like protease. The relative reactivities of amino acid residues in the pr otease-catalyzed hydrolysis of the library can be used as a fingerprint to identify the protease in a protease family.