Chemical Biology of Homocysteine Thiolactone and Related Metabolites

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Abstract

Protein-related homocysteine (Hcy) metabolism produces Hcy-thiolactone, N-Hcy-protein, and Nε-homocysteinyl-lysine (Nε-Hcy-Lys). Hcy-thiolactone is generated in an error-editing reaction in protein biosynthesis when Hcy is erroneously selected in place of methionine by methionyl-tRNA synthetase. Hcy-thiolactone, an intramolecular thioester, is chemically reactive and forms isopeptide bonds with protein lysine residues in a process called N-homocysteinylation, which impairs or alters the

Chemical Synthesis

Hcy-thiolactone can be easily prepared from Met or Hcy. The hydriodic acid digestion procedure, originally developed by Bearnstein for the determination of protein Met content [2], allows quantitative conversion of Met to Hcy-thiolactone with the liberation of methyl iodide. The hydriodic acid digestion of [35S]Met is a convenient method for the preparation of [35S]Hcy-thiolactone for biological studies [5], [6], [7], [12], [32], [33], [34], [35], [36], [37]. Recent studies have shown that the

Synthesis In Vitro

Because the high energy of the anhydride bond of ATP is conserved in the thioester bond of Hcy-thiolactone, it is chemically reactive and easily acylates amino groups in proteins and free-amino acids in vitro forming N-Hcy-protein [5], [6], [7], [27] (Fig. 1). N-Hcy-proteins are easily prepared in vitro by incubating desired protein with Hcy-thiolactone at pH 7.4. The reaction is completed in 4 h at 37 °C or 40 h at 25 °C. When human plasma is incubated with Hcy-thiolactone, each protein becomes N

Chemical Synthesis

The isopeptide Nε-Hcy-Lys (Fig. 1) has been originally identified in vitro as a product of facile reaction of Hcy-thiolactone with lysine [6], [27]. Nε-Hcy-Lys is synthesized in solution phase using common procedures for peptide synthesis starting from commercially available lysine derivative (ε-N-Cbz-Lys-O-Bu) [71]. Nε-Hcy-Lys isopeptide is also synthesized by incubating 5 mmol of l-lysine and 5 mmol of d,l-Hcy-thiolactone hydrochloride [9] in 100 ml 0.2 M sodium phosphate buffer, pH 7.4, 0.2 

Conclusions

As outlined above, protein-related Hcy metabolism generates Hcy-thiolactone, N-Hcy-protein, and Nε-Hcy-Lys. Hcy-thiolactone affects biological function due to its ability to modify protein lysine residues in the process called N-homocysteinylation. N-Homocysteinylation causes protein damage, which is further exacerbated by a thiyl radical-mediated oxidation. N-Hcy-proteins undergo structural changes leading to aggregation and amyloid formation. These structural changes generate proteins, which

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