Kinetics of Isomerization and Inversion of Aspartate 58 of αA-Crystallin Peptide Mimics under Physiological Conditions

Abstract

Although proteins consist exclusively of L-amino acids, we have reported that aspartyl (Asp) 58 and Asp 151 residues of αA-crystallin of eye lenses from elderly cataract donors are highly inverted and isomerized to D-β, D-α and L-β-Asp residues through succinimide intermediates. Of these Asp isomers, large amounts of D-β- and L-β-isomers are present but the amount of D-α-isomer is not significant. The difference in abundance of the Asp isomers in the protein may be due to the rate constants for the formation of the isomers. However, the kinetics have not been well defined. Therefore, in this study, we synthesized a peptide corresponding to human αA-crystallin residues 55 to 65 (T55VLD58SGISEVR65) and its isomers in which L-α-Asp at position 58 was replaced with L-β-, D-β- and D-α-Asp and determined the rate of isomerization and inversion of Asp residues under physiological conditions (37°C, pH7.4). The rate constant for dehydration from L-α-Asp peptide to L-succinimidyl peptide was 3 times higher than the rate constant for dehydration from L-β-Asp peptide to L-succinimidyl peptide. The rate constant for hydrolysis from L-succinimidyl peptide to L-β-Asp peptide was about 5 times higher than the rate constant for hydrolysis from L-succinimidyl peptide to L-α-Asp peptide. The rate constant for dehydration from L-α-Asp peptide to L-succinimidyl peptide was 2 times higher than the rate constant for dehydration from D-α-Asp peptide to D-succinimidyl peptide. The rate constants for hydrolysis from L-succinimidyl peptide to L-β-Asp peptide and for hydrolysis from D-succinimidyl peptide to D-β-Asp peptide were almost equal. Using these rate constants, we calculated the change in the abundance ratios of the 4 Asp isomers during a human lifespan. This result is consistent with the fact that isomerized Asp residues accumulate in proteins during the ageing process.

Racemization of Serine Residues Catalyzed by Dihydrogen Phosphate Ion: A Computational Study

“…This is due to the well-known succinimide-mediated mechanism [12][13][14][15]. In this mechanism, the actual species undergoing racemization is a succinimide intermediate which is formed from an L-Asp residue by an intramolecular cyclization with the loss of a water molecule.…”

Isomerization of aspartyl residues in crystallins and its influence upon cataract

“…Normal L-α-Asp decreases to less than 10% of the total Asp content within the first 5.5 years of life, whereas L-β-Asp markedly increases to 90% and then decreases gradually. D-β-Asp increases gradually, reaching approximately 30% of Asp content at 82 years [36]. Hooi et al previously reported that Asp 151 of αA-crystallin converts to the D-β-, L-β-, and D-α-isomers in a ratio of 3:1:0.5 until 15 years of age; in addition, 40% of Asp 151 is present as the D-β-isomer by 20 years, and this proportion remains fixed until 80 years [39].…”

“…The rate constants k2, k4, k6, k8, k9 and k10 were obtained by heating the L-succinimidyl-and D-succinimidylcontaining peptides at 37°C for 80 min. The rate constants for k1-k10 were calculated by fitting curves of the relative amount of reaction products vs time [36].…”

Staggered side-chain conformers of aspartyl residues prerequisite to transformation from L-α- to D-β-aspartate 58 in human-lens αA-crystallin fragment

“…However, D-aspartyl (Asp) residues have been found in aged lens [1][2][3][4], tooth [5], aorta [6], brain [7,8], bone [9,10], and ligament [11]. D-Asp is produced Biophysical Chemistry 196 (2015) [10][11][12][13][14][15] by the racemization of natural L-Asp residues in the protein. The racemization is accompanied by the isomerization from the natural α-Asp to the uncommon β-Asp form via a succinimide intermediate [12].…”

“…For the racemization, the formation of succinimide is thought to be the rate-determining step [13,14]. Previous studies have suggested that β-Asp is more stable than α-Asp in humanlens αA-crystallin peptides [14].…”