CHEMICAL PATHWAYS OF PEPTIDE DEGRADATION .4. PATHWAYS, KINETICS, AND MECHANISM OF DEGRADATION OF AN ASPARTYL RESIDUE IN A MODEL HEXAPEPTIDE

In this study the hexapeptide Val-Tyr-Pro-Asp-Gly-Ala (Asp-hexapeptide) was used as a model to investigate the kinetics of aspartate degradation in aqueous solution. The apparent rate of degradation of the Asp-hexapeptide was determined as a function of pH, buffer concentration, and temperature. At very acidic pH levels (0.3, 1.1, 1.5, 2.0, and 3.0), the apparent rate of degradation followed pseudo-first-order kinetics. In this pH region, the Asp-hexapeptide predominantly underwent specific acid-catalyzed hydrolysis of the Asp-Gly amide bond (Asp-X hydrolysis) to form a tetrapeptide (Val-Tyr-Pro-Asp) and a dipeptide (Gly-Ala). In addition, parallel formation of a cyclic imide intermediate could be observed, although no isoAsp-hexapeptide was detected. At pH 4.0 and 5.0, the Asp-hexapeptide simultaneously isomerized via the cyclic imide to form the iso-Asp-hexapeptide and underwent Asp-X hydrolysis to produce the cleavage products. The pH-rate profiles (pH 0.3-5.0) for the Asp-X hydrolysis and the formation of cyclic imide revealed that the degree of ionization of the carboxylic acid side chain of Asp residue significantly altered the rate of reaction, with the ionized form being more reactive than the unionized form. Little or no buffer catalysis was observed for either pathway. Solvent isotope experiments were used to probe the mechanism of the Asp-X hydrolysis reaction. At pH values above 6.0, the apparent rate of degradation of the Asp-hexapeptide followed pseudo-first-order reversible kinetics, with the isoAsp-hexapeptide being the only observed product (isomerization). Above pH 8.0, the isomerization kinetics were found to be independent of pH and buffer concentration. The kinetics of degradation of Asp-hexapeptide (Val-Tyr-Pro-Asp-Gly-Ala) and Asn-hexapeptide (Val-Tyr-Pro-Asn-Gly-Ala) were compared to determine the relative instability of the Asp and Asn residues and to understand the mechanism of formation of cyclic imide at near neutral to basic pH.