SUCCINIMIDE-MEDIATED PATHWAY FOR PEPTIDE-BOND CLEAVAGE - KINETIC-STUDY ON AN ASN-SAR CONTAINING PEPTIDE

The cleavage reaction of the peptide bond next to the Asn residue has been studied in the pH range 7.4-13.8 at 37 degrees C and mu = 1M. Thi s reaction yields and N-terminal peptide fragment having at its C-term inus a succinimide ring, which rapidly hydrolyses to both asparagine a nd iso-asparagine residues. For both the two consecutive reactions, pe ptide bond cleavage and the succinimide hydrolysis, the general trend is an increase of the reaction rate with the pH. However, for the hydr olysis reaction there is a small decrease in the pH range 10-11 caused by the deprotonation of the succinimide nitrogen atom. Kinetic eviden ce indicates that the cleavage reaction is a multistep process with a change in the rate-determining step at pH 8.5-9.0. The mechanism invol ves preequilibrium deprotonation of the NH2 amide group of the Asn sid e chain, followed by nucleophilic attack of the nitrogen atom on the c arbonyl carbon atom of the same asparagine residue, giving a cyclic in termediate. Then, general acid-catalyzed departure of the leaving grou p gives the final reaction product. At pH < 8.5, the formation of the cyclic intermediate is rate determining, whereas, at higher pH, it is the departure of the leaving group. (C) 1997 John Wiley & Sons, Inc.