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.

The estimation of glutaminyl deamidation and aspartyl cleavage rates in glucagon.

The major hydrolytic degradation pathways of glucagon under acidic conditions are cleavage at Asp-9, Asp-15, and Asp-21, and deamidation at Gln-3, Gln-20, Gln-24, and Asn-28. The rate constants for these pathways were determined in the pH range 1-2.4 at 60 degrees C by kinetic data analysis of substrate and degradation product concentration-time profiles. Deamidation kinetics were determined using penta-peptide fragments of glucagon containing the labile amide residue. The accurate determination of the cleavage rate constants was confounded by the complexity of the degradation scheme of glucagon. Peptide cleavage kinetics were determined by degradation of glucagon and its cleavage fragments under identical conditions and the use of area-under-the-curve (AUC) and nonlinear regression methods of analysis. Glucagon degradation was first-order with respect to time and concentration in the range of 31-00 microM. Glutaminyl deamidation rate constants were first-order with respect to hydronium ion concentration and were similar for all three residues indicating a lack of sequence effects. The rate constants for Asp cleavage were not first-order with respect to hydronium ion concentration and cleavage at Asp-21 was slower than cleavage at Asp-9 and Asp-15 over the studied pH range.

The relative rates of glutamine and asparagine deamidation in glucagon fragment 22-29 under acidic conditions.

Our objective was to compare the relative rates of asparaginyl and glutaminyl deamidation in fragment 22-29 of the polypeptide hormone glucagon in acidic aqueous solutions. Reaction mixtures containing 22-29 (FVQWLMNT) or its degradation products were degraded at 60 degrees C in dilute hydrochloric acid or phosphate buffer in the pH range 1-3. Degradation products were separated by high-performance liquid chromatography and identified by amino acid sequencing, amino acid analysis, liquid chromatography-mass spectrometry (LC-MS), and matrix-assisted laser desorption and ionization (MALDI). Nine major degradation products were identified, including asparaginyl and glutaminyl deamidated forms, aspartyl peptide cleavage of the asparaginyl deamidated products, and a cyclic imide intermediate. The pH dependences of rate constants for individual pathways were consistent with acid catalysis. Previous investigators have reported a greater susceptibility of asparagine residues to deamidation in neutral and alkaline solutions due to the formation of a more stable five-membered succinimide intermediate. It has been suggested that asparagine may be more labile under acidic conditions also. We have observed a more facile deamidation for the glutamine residue under the acidic condition studied. It is proposed that the lower reactivity of the asparagine residue may be due to a decreased electrophilicity of its side chain carbonyl carbon imparted by a parallel cleavage pathway at this residue.