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Evolutionary importance of the intramolecular pathways of hydrolysis of phosphate ester mixed anhydrides with amino acids and peptides.

Liu Z, Beaufils D, Rossi JC, Pascal R - Sci Rep (2014)

Bottom Line: Even considering CO2 concentrations in early Earth liquid environments equivalent to present levels, mixed anhydrides would have polymerized predominantly through NCAs.The formation of peptide-phosphate mixed anhydrides from 5(4H)-oxazolones (transiently formed through prebiotically relevant peptide activation pathways) was also observed as well as the occurrence of the reverse cyclization process in the reactions of these mixed anhydrides.These processes constitute the core of a reaction network that could potentially have evolved towards the emergence of translation.

View Article: PubMed Central - PubMed

Affiliation: Institut des Biomolécules Max Mousseron, UMR5247 CNRS - University of Montpellier.

ABSTRACT
Aminoacyl adenylates (aa-AMPs) constitute essential intermediates of protein biosynthesis. Their polymerization in aqueous solution has often been claimed as a potential route to abiotic peptides in spite of a highly efficient CO2-promoted pathway of hydrolysis. Here we investigate the efficiency and relevance of this frequently overlooked pathway from model amino acid phosphate mixed anhydrides including aa-AMPs. Its predominance was demonstrated at CO2 concentrations matching that of physiological fluids or that of the present-day ocean, making a direct polymerization pathway unlikely. By contrast, the occurrence of the CO2-promoted pathway was observed to increase the efficiency of peptide bond formation owing to the high reactivity of the N-carboxyanhydride (NCA) intermediate. Even considering CO2 concentrations in early Earth liquid environments equivalent to present levels, mixed anhydrides would have polymerized predominantly through NCAs. The issue of a potential involvement of NCAs as biochemical metabolites could even be raised. The formation of peptide-phosphate mixed anhydrides from 5(4H)-oxazolones (transiently formed through prebiotically relevant peptide activation pathways) was also observed as well as the occurrence of the reverse cyclization process in the reactions of these mixed anhydrides. These processes constitute the core of a reaction network that could potentially have evolved towards the emergence of translation.

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Formation of the dipeptides Ac-Tyr(Me)-Gly-NH2 or H-Tyr(Me)-Gly-NH2 by reaction of 5 mM H-Gly-NH2 with mixed anhydrides 2b or 1b, respectively, in 100 mM pH 6.5 MES buffers.Ratio of the peak area of formed peptides (%): (a) Reaction of 1 mM acyl-aa-PEMA 2b (half-life c.a. 220 min, method A, r.t. 11.43 min, filled squares); (b) Reaction of aa-PEMA 1b in the N2-flushed MES buffer (half-life c.a. 55 min, method A, r.t. 5.85 min, open squares); (c) Reaction of aa-PEMA 1b in the MES buffer to which 2 mM NaHCO3 was added (half-life ≤1 min, method A, r.t. 5.85 min, filled diamonds). The peptide products were identified by ESI-MS.
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f7: Formation of the dipeptides Ac-Tyr(Me)-Gly-NH2 or H-Tyr(Me)-Gly-NH2 by reaction of 5 mM H-Gly-NH2 with mixed anhydrides 2b or 1b, respectively, in 100 mM pH 6.5 MES buffers.Ratio of the peak area of formed peptides (%): (a) Reaction of 1 mM acyl-aa-PEMA 2b (half-life c.a. 220 min, method A, r.t. 11.43 min, filled squares); (b) Reaction of aa-PEMA 1b in the N2-flushed MES buffer (half-life c.a. 55 min, method A, r.t. 5.85 min, open squares); (c) Reaction of aa-PEMA 1b in the MES buffer to which 2 mM NaHCO3 was added (half-life ≤1 min, method A, r.t. 5.85 min, filled diamonds). The peptide products were identified by ESI-MS.

Mentions: At pH 4, the hydrolysis of mixed anhydride 1b was much slower (t1/2 = ca. 550 min) and CO2 catalysis was not observed (Supplementary Information, Fig. S3). This result is consistent with the results obtained by Kluger from alanyl ethyl phosphate24. The protonation of the amino group of 1b increases the electrophilic character of its acyl group and then the rates of nucleophilic attack, but it also prevents any possibility of reaction with CO2 according the pathway of Fig. 3a. The hydrolysis of the acetylated mixed anhydride 2b was indeed observed to be slower (t1/2 ~ 950 min at pH 6.5) and was not affected by addition of 10 mM NaHCO3 (Fig. 6) in a way consistent with this explanation and with previously reported analyses22. However, it is important to emphasize that the CO2-catalyzed pathway does not only constitute a process leading to the deactivation and the hydrolysis of mixed anhydrides since peptide formation can be improved significantly by this means. As a matter of fact, with regard to peptide formation, the prevalence of the NCA pathway was demonstrated by studying the model reaction of 1 mM mixed anhydride 1b with 5 mM glycinamide either in a nitrogen-flushed sample or in the presence of 2 mM NaHCO3 (Fig. 7). Importantly, less than 2 min were sufficient for the starting material to be exhausted in the presence of carbonate, whereas CO2 removal increased the reaction times to much higher values (t1/2 ~ 50 min) and reduced the final yield in dipeptide (Fig. 7). This reaction remained faster than that observed for the acetylated mixed anhydride 2b (t1/2 ~ 260 min) unable to undergo the conversion into NCA, but that will be demonstrated below to partly undergo cyclization into 5(4H)-oxazolones. These experiments carried out using glycinamide for mimicking a growing peptide chain show that the polymerization of adenylates and other aa-PEMA is improved in the presence of CO2 by the occurrence of the NCA pathway owing to both the higher reactivity of the latter intermediate and its ability to suppress diketopiperazine formation.


Evolutionary importance of the intramolecular pathways of hydrolysis of phosphate ester mixed anhydrides with amino acids and peptides.

Liu Z, Beaufils D, Rossi JC, Pascal R - Sci Rep (2014)

Formation of the dipeptides Ac-Tyr(Me)-Gly-NH2 or H-Tyr(Me)-Gly-NH2 by reaction of 5 mM H-Gly-NH2 with mixed anhydrides 2b or 1b, respectively, in 100 mM pH 6.5 MES buffers.Ratio of the peak area of formed peptides (%): (a) Reaction of 1 mM acyl-aa-PEMA 2b (half-life c.a. 220 min, method A, r.t. 11.43 min, filled squares); (b) Reaction of aa-PEMA 1b in the N2-flushed MES buffer (half-life c.a. 55 min, method A, r.t. 5.85 min, open squares); (c) Reaction of aa-PEMA 1b in the MES buffer to which 2 mM NaHCO3 was added (half-life ≤1 min, method A, r.t. 5.85 min, filled diamonds). The peptide products were identified by ESI-MS.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4262824&req=5

f7: Formation of the dipeptides Ac-Tyr(Me)-Gly-NH2 or H-Tyr(Me)-Gly-NH2 by reaction of 5 mM H-Gly-NH2 with mixed anhydrides 2b or 1b, respectively, in 100 mM pH 6.5 MES buffers.Ratio of the peak area of formed peptides (%): (a) Reaction of 1 mM acyl-aa-PEMA 2b (half-life c.a. 220 min, method A, r.t. 11.43 min, filled squares); (b) Reaction of aa-PEMA 1b in the N2-flushed MES buffer (half-life c.a. 55 min, method A, r.t. 5.85 min, open squares); (c) Reaction of aa-PEMA 1b in the MES buffer to which 2 mM NaHCO3 was added (half-life ≤1 min, method A, r.t. 5.85 min, filled diamonds). The peptide products were identified by ESI-MS.
Mentions: At pH 4, the hydrolysis of mixed anhydride 1b was much slower (t1/2 = ca. 550 min) and CO2 catalysis was not observed (Supplementary Information, Fig. S3). This result is consistent with the results obtained by Kluger from alanyl ethyl phosphate24. The protonation of the amino group of 1b increases the electrophilic character of its acyl group and then the rates of nucleophilic attack, but it also prevents any possibility of reaction with CO2 according the pathway of Fig. 3a. The hydrolysis of the acetylated mixed anhydride 2b was indeed observed to be slower (t1/2 ~ 950 min at pH 6.5) and was not affected by addition of 10 mM NaHCO3 (Fig. 6) in a way consistent with this explanation and with previously reported analyses22. However, it is important to emphasize that the CO2-catalyzed pathway does not only constitute a process leading to the deactivation and the hydrolysis of mixed anhydrides since peptide formation can be improved significantly by this means. As a matter of fact, with regard to peptide formation, the prevalence of the NCA pathway was demonstrated by studying the model reaction of 1 mM mixed anhydride 1b with 5 mM glycinamide either in a nitrogen-flushed sample or in the presence of 2 mM NaHCO3 (Fig. 7). Importantly, less than 2 min were sufficient for the starting material to be exhausted in the presence of carbonate, whereas CO2 removal increased the reaction times to much higher values (t1/2 ~ 50 min) and reduced the final yield in dipeptide (Fig. 7). This reaction remained faster than that observed for the acetylated mixed anhydride 2b (t1/2 ~ 260 min) unable to undergo the conversion into NCA, but that will be demonstrated below to partly undergo cyclization into 5(4H)-oxazolones. These experiments carried out using glycinamide for mimicking a growing peptide chain show that the polymerization of adenylates and other aa-PEMA is improved in the presence of CO2 by the occurrence of the NCA pathway owing to both the higher reactivity of the latter intermediate and its ability to suppress diketopiperazine formation.

Bottom Line: Even considering CO2 concentrations in early Earth liquid environments equivalent to present levels, mixed anhydrides would have polymerized predominantly through NCAs.The formation of peptide-phosphate mixed anhydrides from 5(4H)-oxazolones (transiently formed through prebiotically relevant peptide activation pathways) was also observed as well as the occurrence of the reverse cyclization process in the reactions of these mixed anhydrides.These processes constitute the core of a reaction network that could potentially have evolved towards the emergence of translation.

View Article: PubMed Central - PubMed

Affiliation: Institut des Biomolécules Max Mousseron, UMR5247 CNRS - University of Montpellier.

ABSTRACT
Aminoacyl adenylates (aa-AMPs) constitute essential intermediates of protein biosynthesis. Their polymerization in aqueous solution has often been claimed as a potential route to abiotic peptides in spite of a highly efficient CO2-promoted pathway of hydrolysis. Here we investigate the efficiency and relevance of this frequently overlooked pathway from model amino acid phosphate mixed anhydrides including aa-AMPs. Its predominance was demonstrated at CO2 concentrations matching that of physiological fluids or that of the present-day ocean, making a direct polymerization pathway unlikely. By contrast, the occurrence of the CO2-promoted pathway was observed to increase the efficiency of peptide bond formation owing to the high reactivity of the N-carboxyanhydride (NCA) intermediate. Even considering CO2 concentrations in early Earth liquid environments equivalent to present levels, mixed anhydrides would have polymerized predominantly through NCAs. The issue of a potential involvement of NCAs as biochemical metabolites could even be raised. The formation of peptide-phosphate mixed anhydrides from 5(4H)-oxazolones (transiently formed through prebiotically relevant peptide activation pathways) was also observed as well as the occurrence of the reverse cyclization process in the reactions of these mixed anhydrides. These processes constitute the core of a reaction network that could potentially have evolved towards the emergence of translation.

Show MeSH