The effects of a histidine residue on the C-terminal side of an asparaginyl residue on the rate of deamidation using model pentapeptides.

The effects of a histidine (His) residue located on the C-terminal side of an asparaginyl (Asn) residue on the rate of deamidation were studied using Gly-Gln-Asn-X-His pentapeptides. The rates of deamidation of the pentapeptides were determined at 37 degrees C (I = 0.5) as function of pH, buffer species, and buffer concentration. A capillary electrophoresis stability-indicating assay was developed to monitor simultaneously the disappearance of the starting peptides and the appearance of the degradation products. The rates of degradation of the peptides were pH dependent, increasing with pH, and followed apparent first-order kinetics. At pH values <6.5, Gly-Gln-Asn-His-His degraded faster than Gly-Gln-Asn-Gly-His, suggesting that the N+1 His residue is catalyzing the deamidation of the Asn residue. The His side chain at these pH values could function as a general acid catalyst, stabilizing the oxyanionic transition state of the cyclic imide intermediate formation. In contrast, at pH values >6.5, Gly-Gln-Asn-Gly-His deamidates more rapidly than Gly-Gln-Asn-His-His. The bulk of the side chain of the N+1 His residue versus the N+1 Gly residue apparently inhibits the flexibility of the peptide around the reaction site and, consequently, reduces the rate of the reaction. The significance of this steric hindrance effect of the N+1 His residue on the rate of deamidation was examined further. It was observed that at pH >6.0, Gly-Gln-Asn-His-His undergoes deamidation faster than Gly-Gln-Asn-Val-His. This observation indicated that, at the higher pH values, the N+1 His residue is also acting as a catalyst. Thus, at basic pH, the N+1 His residue influences the rate of deamidation via two opposing effects; that is, general base catalysis and steric interference. The pentapeptide Gly-Gln-Asn-His-His, in addition to undergoing the deamidation reaction, also undergoes bond cleavage at the Asn-His peptide bond. The enhanced rate of Asn-His peptide bond cleavage can be attributed to the general base behavior of the His residue, leading to increased nucleophilicity of the Asn side-chain amide group. Finally, we have shown that the His residue that is two amino acids removed from the Asn, the N+2 position, has little or no effect on the rate of deamidation.

Comparison of the rates of deamidation, diketopiperazine formation and oxidation in recombinant human vascular endothelial growth factor and model peptides.

In this work, we examine the way in which stability information obtained from studies on small model peptides correlates with similar information acquired from a protein. The rates of deamidation, oxidation, and diketopiperazine reactions in model peptide systems were compared to those of recombinant human vascular endothelial growth factor (rhVEGF). The N-terminal residues of rhVEGF, a potent mitogen in angiogenesis, are susceptible to the aforementioned reactions. The degradation of the peptides L-Ala-L-Pro-L-Met (APM) and Gly-L-Gln-L-Asn-L-His-L-His (GQNHH), residues 1-3 and 8-12 of rhVEGF, respectively, and rhVEGF were examined at pH 5 and 8 at 37 degrees C. Capillary electrophoresis and high-performance liquid chromatography (HPLC) stability-indicating assays were developed to monitor the degradation of the penta- and tripeptides, respectively. The degradation of rhVEGF was determined by tryptic mapping and quantified by RP-HPLC. The rates of degradation of both peptides and the protein followed apparent first-order kinetics and increased with increasing pH. The tripeptide APM underwent diketopiperazine formation (Ala-Pro-diketopiperazine) and oxidation of the Met residue, whereas the pentapeptide GQNHH degraded via the deamidation pathway. The results indicate that the rates of deamidation and oxidation of the protein are comparable to those observed in the model peptides at both pH values. However, the rate of the diketo-piperazine reaction was slower in the protein than in the model peptide, which may be the result of differences in the cis-trans equilibrium of the X-Pro peptide bonds in the 2 molecules.

Kinetics of diketopiperazine formation using model peptides.

The intramolecular aminolysis of Phe-Pro-p-nitroaniline (Phe-Pro-pNA) to Phe-Pro-diketopiperazine (Phe-Pro-DKP) was studied as a function of pH, temperature, buffer concentration, and buffer species using an HPLC assay that permits simultaneous analysis of the disappearance of the starting material and the appearance of degradation products. The degradation followed pseudo-first-order kinetics and showed significant dependence on pH. Phosphate (pH 5-8) and glycine (pH 9-10) buffers exhibit general base catalysis. The pH-rate profile suggested that the rate of Phe-Pro-DKP formation depends on the degree of ionization of the N-terminal amino group, with the unprotonated reactant being more reactive than the protonated form. The pKa value of 6.1, determined kinetically, and three microscopic rate constants were adequate to describe the shape of the pH-rate profile. In the pH range studied, Phe-Pro-DKP was the only product generated upon degradation of Phe-Pro-pNA. At pH values between 3 and 8, Phe-Pro-DKP was stable, while at pH less than 3 and greater than 8 it undergoes hydrolysis to the dipeptide, Phe-Pro-OH. Sequence inversion, a reaction normally associated with DKP formation, was not observed. The influence of primary sequence on the formation of DKP was also investigated using X-Pro-pNA analogues, where X = Gly, Ala, Val, Phe, beta-cyclohexylalanine, and Arg. Changing the amino acid preceding the proline residue had a significant effect on the rate of DKP formation at pH 7.0.