Neighboring Side Chain Effects on Asparaginyl and Aspartyl Degradation:  An Ab Initio Study of the Relationship between Peptide Conformation and Backbone NH Acidity

Abstract

The rate of spontaneous degradations of asparagine and aspartyl residues occurring through succinimide intermediates is dependent upon the nature of the residue on the carboxyl side in peptides. For nonglycine residues, we show here that this effect can largely be attributed to the electrostatic/inductive effect of the side chain group on the equilibrium concentration of the anionic form of the peptide bond nitrogen atom that initiates the succinimide forming reaction. However, the rate of degradation of Asn-Gly and Asp-Gly containing peptides is about an order of magnitude greater than predicted solely using this explanation. To understand the nature of the glycine effect, ab initio calculations were performed on model compounds. These calculations indicate that there is little to no change in the stability of the transition state or the tetrahedral intermediate of succinimide formation with Asn-/Asp-Gly and Asn-/Asp-Ala derivatives. However, we have found that the acidity of the backbone peptide nitrogen NH is highly dependent upon the conformation of the molecule. Since glycine residues lack the beta-carbon common to all other protein amino acids, these residues can sample additional regions of conformational space where it is possible to further stabilize the backbone amide anion and thus increase the rate of degradation. These results provide the first rationale for the particular rate enhancement of degradation in peptidyl Asn-/Asp-Gly sequences. The results also can be applied to asparagine and aspartyl residues in proteins where the 3-dimensional structure provides additional constraints on conformation that can either increase or decrease the equilibrium concentration of the backbone amide anion and thus their rate of degradation via succinimide intermediates. Understanding this chemistry will assist attempts to minimize the deleterious effect of aging at the molecular level. The relationship between these results and proton exchange experiments is discussed in the Appendix.

Racemisation and human cataract. d-Ser, d-Asp/Asn and d-Thr are higher in the lifelong proteins of cataract lenses than in age-matched normal lenses

“…Asp 151 in alpha A crystallin, which has been identified by Fujii's group as being particularly susceptible to racemisation in human lenses (Fujii et al 1999b) may be one such site, since our initial data suggest that it is approximately 25% racemized by age 20. Conformationally, flexible regions, those in the terminal regions of proteins (Di Salvo et al 1999) and those that contain favourable sequences, such as Asx-Gly, may be particularly susceptible to inversion (Radkiewicz et al 2001).…”

FlhB Regulates Ordered Export of Flagellar Components via Autocleavage Mechanism

“…It is thought that different conformations induced by changes in the side-chain of the residue following Asn make the peptide nitrogen more or less acidic (thus affecting the equilibrium between the protonated and the deprotonated states) (34). It is thus not unexpected that the replacement of the Pro residue by Ala in the FlhB(P270A) variant resulted in an altered cleavage behavior from that of wild-type FlhB.…”

Protein Repair Methyltransferase from the Hyperthermophilic Archaeon Pyrococcus furiosus

“…Protein L-isoaspartyl-(D-aspartyl) O-methyltransferase (EC 2.1.1.77) is a repair enzyme that catalyzes the S-adenosylmethionine (AdoMet)-dependent 1 methyl esterification of the ␣-carboxyl group of L-isoaspartyl residues that originate from the spontaneous degradation of aspartic acid and asparagine residues in proteins (1)(2)(3)(4)(5). The enzyme-mediated methylation reaction is followed by nonenzymatic steps that result in the net conversion of L-isoaspartyl residues to L-aspartyl residues, representing a potentially important mechanism for avoiding the accumulation of damaged proteins as cells age (4 -9).…”