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. This reaction yields and N-terminal peptide fragment having at its C-terminus a succinimide ring, which rapidly hydrolyses to both asparagine and iso-asparagine residues. For both the two consecutive reactions, peptide bond cleavage and the succinimide hydrolysis, the general trend is an increase of the reaction rate with the pH. However, for the hydrolysis reaction there is a small decrease in the pH range 10-11 caused by the deprotonation of the succinimide nitrogen atom. Kinetic evidence 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 involves preequilibrium deprotonation of the NH2 amide group of the Asn side chain, followed by nucleophilic attack of the nitrogen atom on the carbonyl carbon atom of the same asparagine residue, giving a cyclic intermediate. Then, general acid-catalyzed departure of the leaving group 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.