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

本项目旨在建立一种新的系统分析内肽酶特异性的方法。为此，我们制备了树脂连接的肽库，其中只有一个特定的位置被各种氨基酸残基占据，并使用这些库作为蛋白酶的底物。通过Edman降解的自动肽测序定量分析水解产物。1）我们对各种树脂进行了比较，发现氨甲基树脂最适合作为合成树脂结合肽库的固相载体。以这些作为底物，我们研究了间隔区对切割效率的影响，发现五甘氨酸作为间隔区是足够的，其使胰凝乳蛋白酶的切割效率增加约30倍。2）通过Fmoc方法使用Fmoc-氨基酸在一个特定位置的等摩尔混合物的肽合成没有得到所需肽的等摩尔混合物，各肽的产率相差10倍。通过改变Fmoc-氨基酸在混合物中的相对摩尔比克服了这一点;因此，对于大多数氨基酸，产率的差异可以减小到3倍以内。3）我们通过将其应用于胰凝乳蛋白酶亚位点特异性的研究来检验这种方法的有用性。作为模型肽库，我们制备了Arg-Pro-Xxx-Phe-Ser-Pro-Arg（Gly）_5-Resin、N-acetyl-Arg-Pro-Gly-Phe-Xxx-Pro-Arg-（Gly）_5-Resin和N-acetyl-Arg-Pro-Gly-Phe-Ser-Xxx-Arg-（Gly）_5-Resin，其中Xxx是各种氨基酸的混合物，将它们进行胰凝乳蛋白酶水解并分析水解产物。这些结果为我们研究胰凝乳蛋白酶的P_2-、P_1 '-和P_2'-位点特异性提供了新的有趣的信息。

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.