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 pentapepti des were determined at 37 degrees C (I = 0.5) as function of pH, buffer spe cies, and buffer concentration. A capillary electrophoresis stability-indic ating assay was developed to monitor simultaneously the disappearance of th e starting peptides and the appearance of the degradation products. The rat es of degradation of the peptides were pH dependent, increasing with pH, an d followed apparent first-order kinetics. At pH values <6.5, Gly-Gln-Asn-Hi s-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 cha in at these pH values could function as a general acid catalyst, stabilizin g the oxyanionic transition state of the cyclic imide intermediate formatio n. In contrast, at pH values >6.5, Gly-Gln-Asn-Gly-His deamidates more rapi dly than Gly-Gln-Asn-His-His. The bulk of the side chain of the N+1 His res idue 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 th at at pH >6.0, Gly-Gln-Asn-His-His undergoes deamidation faster than Gly-Gl n-Asn-Val-His. This observation indicated that, at the higher pH values, th e 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; th at 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 A sn-His peptide bond cleavage can be attributed to the general base behavior of the His residue, leading to increased nucleophilicity of the Asn side-c hain amide group. Finally, we have shown that the His residue that is two a mino acids removed from the Asn, the N+2 position, has little or no effect on the rate of deamidation. (C) 2000 Wiley-Liss, Inc.