Engineering of substrate mimetics as novel-type substrates for glutamic acid-specific endopeptidases: design, synthesis, and application

This account reports on the development and function of novel substrate mim etics as artificial substrates for Glu-specific endopeptidases. Firstly, in an empirical way, various aliphatic and aromatic analogs of the already es tablished carboxymethyl thioester-substrate mimetics were designed from sim ple structure-function relationship studies. The specificity of the newly d eveloped substrates for Staphylococcus aureus V8 protease-catalyzed reactio ns have been examined by steady-state hydrolysis kinetic studies. Additiona lly, these studies were expanded to the use of the equally Glu-specific end opeptidase from Bacillus licheniformis (BL-GSE) which can easily be purifie d from alcalase in high yields. Finally, the novel substrate mimetics were used as acyl donor components in BL-GSE- and V8 protease-catalyzed model ac yl transfer reactions. The results clarify the newly developed substrate mi metics as efficient acyl donors as well as BL-GSE as an attractive alternat ive to V8 protease for enzymatic peptide synthesis. (C) 2000 Elsevier Scien ce B.V. All rights reserved.