Enzymatic coupling of specific peptides at nonspecific ligation sites: Effect of Asp189Glu mutation in trypsin on substrate mimetic-mediated reactions

Two main drawbacks seriously restrict the synthetic value of proteases as r eagents in peptide fragment coupling: (i) native proteolytic activity and, thus, risk of undesired peptide cleavage; (ii) limited enzyme specificities restricting the amino acid residues between which a peptide bond can be fo rmed. While the latter can be overcome by the use of substrate mimetics ach ieving peptide bond formation at nonspecific ligation sites, the risk of pr oteolytic cleavage still remains and hinders the wide acceptance of this po werful strategy for peptide coupling. This paper reports on the effect of t he trypsin point mutant Asp189Glu on substrate mimetic-mediated reactions. The effect of this mutation on the steady-state hydrolysis of substrate mim etics of the 4-guanidinophenyl ester type and on trypsin-specific Lys- and Arg-containing peptides was investigated. The results were confirmed by enz ymatic coupling reactions using substrate mimetics as the acyl donor and sp ecific amino acid-containing peptides as the acyl acceptor. The competition assay verifies the predicted shift, in substrate preference from Lys and A rg to the substrate mimetics and, thus, from cleavage to synthesis of pepti de bonds. The combination of results obtained qualifies the trypsin mutant D189E as the first substrate mimetic-specific peptide ligase.