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Stereochemical course of hydrolytic reaction catalyzed by alpha-galactosidase from cold adaptable marine bacterium of genus Pseudoalteromonas.

Bakunina IY, Balabanova LA, Golotin VA, Slepchenko LV, Isakov VV, Rasskazov VA - Front Chem (2014)

Bottom Line: In order to understand the mechanism of action, the stereochemistry of hydrolysis of 4-nitrophenyl α-D-galactopyranoside (4-NPGP) by α-PsGal was measured by (1)H NMR spectroscopy.The kinetics of formation of α- and β-anomer of galactose showed that α-anomer initially formed and accumulated, and then an appreciable amount of β-anomer appeared as a result of mutarotation.The data clearly show that the enzymatic hydrolysis of 4-NPGP proceeds with the retention of anomeric configuration, probably, due to a double displacement mechanism of reaction.

View Article: PubMed Central - PubMed

Affiliation: G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences Vladivostok, Russia.

ABSTRACT
The recombinant α-galactosidase of the marine bacterium (α-PsGal) was synthesized with the use of the plasmid 40Gal, consisting of plasmid pET-40b (+) (Novagen) and the gene corresponding to the open reading frame of the mature α-galactosidase of marine bacterium Pseudoalteromonas sp. KMM 701, transformed into the Escherichia coli Rosetta(DE3) cells. In order to understand the mechanism of action, the stereochemistry of hydrolysis of 4-nitrophenyl α-D-galactopyranoside (4-NPGP) by α-PsGal was measured by (1)H NMR spectroscopy. The kinetics of formation of α- and β-anomer of galactose showed that α-anomer initially formed and accumulated, and then an appreciable amount of β-anomer appeared as a result of mutarotation. The data clearly show that the enzymatic hydrolysis of 4-NPGP proceeds with the retention of anomeric configuration, probably, due to a double displacement mechanism of reaction.

No MeSH data available.


Related in: MedlinePlus

1H NMR analysis of the stereo selectivity for the recombinant α-PsGal catalyzed reaction. One-dimensional 1H NMR spectra of the deuterium-exchanged 4-NPGP prior to enzyme addition, at τ = 0 min (spectrum 1), at τ = 3 min (spectrum 2); at τ = 6 min (spectrum 3); at τ = 24 min (spectrum 4); at τ = 60 min (spectrum 5) after initiation of the reaction. (in 50 mM sodium phosphate buffer pH 7.5, at 20°C, D2O).
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Figure 3: 1H NMR analysis of the stereo selectivity for the recombinant α-PsGal catalyzed reaction. One-dimensional 1H NMR spectra of the deuterium-exchanged 4-NPGP prior to enzyme addition, at τ = 0 min (spectrum 1), at τ = 3 min (spectrum 2); at τ = 6 min (spectrum 3); at τ = 24 min (spectrum 4); at τ = 60 min (spectrum 5) after initiation of the reaction. (in 50 mM sodium phosphate buffer pH 7.5, at 20°C, D2O).

Mentions: 1H NMR spectra of deuterium-exchanged 4-NPGP and products of it's hydrolysis under the action of the recombinant α-PsGal are shown on Figure 3.


Stereochemical course of hydrolytic reaction catalyzed by alpha-galactosidase from cold adaptable marine bacterium of genus Pseudoalteromonas.

Bakunina IY, Balabanova LA, Golotin VA, Slepchenko LV, Isakov VV, Rasskazov VA - Front Chem (2014)

1H NMR analysis of the stereo selectivity for the recombinant α-PsGal catalyzed reaction. One-dimensional 1H NMR spectra of the deuterium-exchanged 4-NPGP prior to enzyme addition, at τ = 0 min (spectrum 1), at τ = 3 min (spectrum 2); at τ = 6 min (spectrum 3); at τ = 24 min (spectrum 4); at τ = 60 min (spectrum 5) after initiation of the reaction. (in 50 mM sodium phosphate buffer pH 7.5, at 20°C, D2O).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4195319&req=5

Figure 3: 1H NMR analysis of the stereo selectivity for the recombinant α-PsGal catalyzed reaction. One-dimensional 1H NMR spectra of the deuterium-exchanged 4-NPGP prior to enzyme addition, at τ = 0 min (spectrum 1), at τ = 3 min (spectrum 2); at τ = 6 min (spectrum 3); at τ = 24 min (spectrum 4); at τ = 60 min (spectrum 5) after initiation of the reaction. (in 50 mM sodium phosphate buffer pH 7.5, at 20°C, D2O).
Mentions: 1H NMR spectra of deuterium-exchanged 4-NPGP and products of it's hydrolysis under the action of the recombinant α-PsGal are shown on Figure 3.

Bottom Line: In order to understand the mechanism of action, the stereochemistry of hydrolysis of 4-nitrophenyl α-D-galactopyranoside (4-NPGP) by α-PsGal was measured by (1)H NMR spectroscopy.The kinetics of formation of α- and β-anomer of galactose showed that α-anomer initially formed and accumulated, and then an appreciable amount of β-anomer appeared as a result of mutarotation.The data clearly show that the enzymatic hydrolysis of 4-NPGP proceeds with the retention of anomeric configuration, probably, due to a double displacement mechanism of reaction.

View Article: PubMed Central - PubMed

Affiliation: G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences Vladivostok, Russia.

ABSTRACT
The recombinant α-galactosidase of the marine bacterium (α-PsGal) was synthesized with the use of the plasmid 40Gal, consisting of plasmid pET-40b (+) (Novagen) and the gene corresponding to the open reading frame of the mature α-galactosidase of marine bacterium Pseudoalteromonas sp. KMM 701, transformed into the Escherichia coli Rosetta(DE3) cells. In order to understand the mechanism of action, the stereochemistry of hydrolysis of 4-nitrophenyl α-D-galactopyranoside (4-NPGP) by α-PsGal was measured by (1)H NMR spectroscopy. The kinetics of formation of α- and β-anomer of galactose showed that α-anomer initially formed and accumulated, and then an appreciable amount of β-anomer appeared as a result of mutarotation. The data clearly show that the enzymatic hydrolysis of 4-NPGP proceeds with the retention of anomeric configuration, probably, due to a double displacement mechanism of reaction.

No MeSH data available.


Related in: MedlinePlus