Discovery of Intermediates of lacZ β-Galactosidase Catalyzed Hydrolysis Using dDNP NMR

Christian Kjeldsen, Jan Henrik Ardenkjær-Larsen, Jens Ø. Duus*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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Abstract

Using dissolution dynamic nuclear polarization, the sensitivity of single scan solution state 13C NMR can be improved up to 4 orders of magnitude. In this study, the enzyme lacZ β-galactosidase from Escherichia coli was subjected to hyperpolarized substrate, and previously unknown reaction intermediates were observed, including a 1,1-linked disaccharide. The enzyme is known for making 1,6-transglycosylation, producing products like allolactose, that are also substrates. To analyze the kinetics, a simple kinetic model was developed and used to determine relative transglycosylation and hydrolysis rates of each of the intermediates, and the novel transglycosylation intermediates were determined as better substrates than the 1,6-linked one, explaining their transient nature. These findings suggest that hydrolysis and transglycosylation might be more complex than previously described.
Original languageEnglish
JournalJournal of the American Chemical Society
Volume140
Issue number8
Pages (from-to)3030-3034
ISSN0002-7863
DOIs
Publication statusPublished - 2018

Cite this

@article{07ded81b0a3f4c2d914b4fe05758d107,
title = "Discovery of Intermediates of lacZ β-Galactosidase Catalyzed Hydrolysis Using dDNP NMR",
abstract = "Using dissolution dynamic nuclear polarization, the sensitivity of single scan solution state 13C NMR can be improved up to 4 orders of magnitude. In this study, the enzyme lacZ β-galactosidase from Escherichia coli was subjected to hyperpolarized substrate, and previously unknown reaction intermediates were observed, including a 1,1-linked disaccharide. The enzyme is known for making 1,6-transglycosylation, producing products like allolactose, that are also substrates. To analyze the kinetics, a simple kinetic model was developed and used to determine relative transglycosylation and hydrolysis rates of each of the intermediates, and the novel transglycosylation intermediates were determined as better substrates than the 1,6-linked one, explaining their transient nature. These findings suggest that hydrolysis and transglycosylation might be more complex than previously described.",
author = "Christian Kjeldsen and Ardenkj{\ae}r-Larsen, {Jan Henrik} and Duus, {Jens {\O}.}",
year = "2018",
doi = "10.1021/jacs.7b13358",
language = "English",
volume = "140",
pages = "3030--3034",
journal = "Journal of the American Chemical Society",
issn = "0002-7863",
publisher = "ACS Publications",
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}

Discovery of Intermediates of lacZ β-Galactosidase Catalyzed Hydrolysis Using dDNP NMR. / Kjeldsen, Christian; Ardenkjær-Larsen, Jan Henrik; Duus, Jens Ø.

In: Journal of the American Chemical Society, Vol. 140, No. 8, 2018, p. 3030-3034.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Discovery of Intermediates of lacZ β-Galactosidase Catalyzed Hydrolysis Using dDNP NMR

AU - Kjeldsen, Christian

AU - Ardenkjær-Larsen, Jan Henrik

AU - Duus, Jens Ø.

PY - 2018

Y1 - 2018

N2 - Using dissolution dynamic nuclear polarization, the sensitivity of single scan solution state 13C NMR can be improved up to 4 orders of magnitude. In this study, the enzyme lacZ β-galactosidase from Escherichia coli was subjected to hyperpolarized substrate, and previously unknown reaction intermediates were observed, including a 1,1-linked disaccharide. The enzyme is known for making 1,6-transglycosylation, producing products like allolactose, that are also substrates. To analyze the kinetics, a simple kinetic model was developed and used to determine relative transglycosylation and hydrolysis rates of each of the intermediates, and the novel transglycosylation intermediates were determined as better substrates than the 1,6-linked one, explaining their transient nature. These findings suggest that hydrolysis and transglycosylation might be more complex than previously described.

AB - Using dissolution dynamic nuclear polarization, the sensitivity of single scan solution state 13C NMR can be improved up to 4 orders of magnitude. In this study, the enzyme lacZ β-galactosidase from Escherichia coli was subjected to hyperpolarized substrate, and previously unknown reaction intermediates were observed, including a 1,1-linked disaccharide. The enzyme is known for making 1,6-transglycosylation, producing products like allolactose, that are also substrates. To analyze the kinetics, a simple kinetic model was developed and used to determine relative transglycosylation and hydrolysis rates of each of the intermediates, and the novel transglycosylation intermediates were determined as better substrates than the 1,6-linked one, explaining their transient nature. These findings suggest that hydrolysis and transglycosylation might be more complex than previously described.

U2 - 10.1021/jacs.7b13358

DO - 10.1021/jacs.7b13358

M3 - Journal article

VL - 140

SP - 3030

EP - 3034

JO - Journal of the American Chemical Society

JF - Journal of the American Chemical Society

SN - 0002-7863

IS - 8

ER -