Development of a systematic method for analyzing the specificity of proteases and its application

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This project aimed to develop a new systematic method for analyzing the specificity of endopeptidases. For this purpose, we prepared resin-linked peptide libraries, in which only one specific position was occupied with various amino acid residues, and used these libraries as substrates for a protease. The hydrolysis products were analyzed quantitatively by automated peptide sequencing by Edman degradation. The results demonstrated the usefulness of this method.1) We checked various resins and found that an aminomethyl-resin was most suitable as the solid support for synthesis of resin-bound peptide libraries. With these as substrates, we investigated the effect of spacers on the cleavage efficiency and found that pentaglycine was sufficient as a spacer, which increased the cleavage efficiency by chymotrypsin about 30-fold.2) The peptide synthesis by the Fmoc-method using an equimolar mixture of Fmoc-amino acids at one specific position did not give an equimolar mixture of the desired peptides, a 10-times difference being obtained in the yields of respective peptides. This was overcome by changing the relative molar ratio of the Fmoc-amino acids in the mixture ; thus the difference in the yield could be reduced to within 3-fold for most amino acids.3) We examined the usefulness of this method by applying it to study on the subsite specificities of chymotrypsin. As model peptide libraries, we prepared Arg-Pro-Xxx-Phe-Ser-Pro-Arg (Gly)_5-Resin, N-acetyl-Arg-Pro-Gly-Phe-Xxx-Pro-Arg- (Gly)_5-Resin and N-acetyl-Arg-Pro-Gly-Phe-Ser-Xxx-Arg- (Gly)_5-Resin, where Xxx was a mixture of various amino acids, submitted them to chymotryptic hydrolysis and analyzed the hydrolysis products. The results provided us with novel and interesting information on the P_2-, P_1'-, and P_2'-site specificities of chymotrypsin.4) We purified various novel proteases to be examined by the present methods.