Studies on the mechanism of aspartic acid cleavage and glutamine deamidation in the acidic degradation of glucagon

In this study, the polypeptide hormone glucagon was used as a model to investigate the mechanisms of aspartic acid cleavage and glutaminyl deamidation in acidic aqueous solutions. Kinetic studies have shown that cleavage at Asp-21 occurred at significantly slower rates than at Asp-9 and Asp-15 while deamidation rates were similar at the three Gln residues. The role of side-chain ionization in the cleavage mechanism was investigated by determining the pK(a) values of the three Asp residues using TOCSY and NOESY NMR methods. The role of proton transfer was investigated using kinetic solvent isotope effect studies (KSIE). The pK(a) values for the sidechains of Asp-9, Asp-15, and Asp-21 were found to be 3.69, 3.72, and 4.05 respectively. No kinetic solvent isotope effect was observed for the cleavage reaction whereas an inverse effect was observed for deamidation. Based on the lack of sequence effects, pH-rate behavior, and KSIE, the deamidation mechanism was proposed to involve direct hydrolysis of the amide side-chain by water. Based on substrate ionization, pH-rate profiles, and KSIE, the proposed mechanism for Asp cleavage involved nucleophilic attack of the ionized side-chain carboxylate on the protonated carbonyl carbon of the peptide bond to give a cyclic anhydride intermediate. (c) 2005 Wiley-Liss, Inc. and the American Pharmacists Association.