Radical acylation of L-lysine derivatives and L-lysine-containing peptides by peroxynitrite-treated diacetyl and methylglyoxal

Abstract

Highly electrophilic α-dicarbonyls such as diacetyl, methylglyoxal, 3-deoxyglucosone, and4,5-dioxovaleric acid have been characterized as secondary catabolites that can aggregate proteins and form DNA nucleobase adducts in several human maladies, including Alzheimer's disease, rheumatoid arthritis, diabetes, sepsis, renal failure, and respiratory distress syndrome. In vitro, diacetyl and methylglyoxal have also been shown to rapidly add up the peroxynitrite anion (k2 ~ 10(4)-10(5) M(-1) s(-1)), a potent biological nucleophile, oxidant and nitrosating agent, followed by carbon chain cleavage to carboxylic acids via acetyl radical intermediate that can modify amino acids. In this study, we used the amino acid derivatives Ac-Lys-OMe and Z-Lys-OMe and synthesized the tetrapeptides H-KALA-OH, Ac-KALA-OH, and H-K(Boc)ALA-OH to reveal the preferential Lys amino group targeted by acyl radical generated by the α-dicarbonyl/peroxynitrite system. The pH profiles of the reactions are bell-shaped, peaking at approximately 7.5; hence, they are close to the pKa values of ONOOH and of the catalytic H2PO4(-) anion. RP-HPLC and ESI-MS analyses of reaction products confirmed (α)N- and (ϵ)N-acetylation of Lys by diacetyl as well as acetylation and formylation by methylglyoxal, with preference for the α-amino group. These data suggest the possibility of radical acylation of proteins in epigenetic processes, where enzymatic acetylation of these biomolecules is a well-documented event, recently reported to be as critical to the cell cycle as phosphorylation. Also noteworthy is the observed formylation of L-Lys containing peptides by methylglyoxal never reported to occur in amino acid residues of peptides and proteins.

Increased chemical acetylation of peptides and proteins in rats after daily ingestion of diacetyl analyzed by Nano-Lc-MS/MS

“…[33] The increase in chemical acetylation of lung proteins of diacetyl-treated rats described here may be connected with the fact that diacetyl has been shown in vitro to generate acetyl radicals upon reaction with peroxynitrite, and more slowly with hydrogen peroxide. [13] The diacetyl/peroxynitrite system was then reported to promote acetylation of isolated aminoacids, peptides and albumin. These data led us to postulate that post-translational chemical acetylation of proteins may contribute to enzymatic acetylation at sites where both diacetyl and peroxynitrite at inflammation are formed.…”

“…[5,8,9] Recently, we reported that the reaction of peroxynitrite with α-dicarbonyls, namely diacetyl and methylglyoxal, in aerated phosphate buffer pH 7.4 results in the acetylation of free amino acids, peptides and proteins added to the reaction mixture. [10][11][12][13] This reaction is initiated by nucleophilic addition of peroxynitrite to the carbonyl group of the α-dicarbonyl compound yielding a peroxynitroso adduct, whose homolysis yields acetyl radicals. Dissolved molecular oxygen adds to the radical to ultimately produce acetate from diacetyl or acetate and formate from methylglyoxal.…”

“…[11][12] Formyl radical intermediate generated by methylglyoxal/peroxynitrite was shown to add to the α-amino group of L-lysine-containing synthesized tetrapeptides. [13] On the other hand, diacetyl/peroxynitrite-generated acetyl radicals have proven been proven to attack both the α-and ε-amino groups of free and blocked L-Lys, L-Lys-containing peptides and serum albumin. [10] These findings have raised the hypothesis that radical acetylation of proteins contributes to transacetylase-promoted post-translational protein modifications at sites where both methylglyoxal or diacetyl and peroxynitrite are present.…”

“…[10] These findings have raised the hypothesis that radical acetylation of proteins contributes to transacetylase-promoted post-translational protein modifications at sites where both methylglyoxal or diacetyl and peroxynitrite are present. [10][11][12][13] From these facts, the competition of chemical (induced by diacetyl) and enzymatic (occurring naturally in organisms) acetylation can be inferred, with the former process contributing to the increase of total protein acetylation.…”

Mecanismos da reação de metabólitos α-dicarbonílicos com peroxinitrito: geração de radicais livres e oxigênio singlete. Possíveis implicações biológicas

“…Mecanismo Hipotético para a Reação de Peroxinitrito com Metilglioxal. Pelo fato de aldeídos serem mais reativos do que cetonas, espera-se que a rota 1 seja predominante 99 .…”