NMR studies of protonation and hydrogen bond states of internal aldimines of pyridoxal 5′-phosphate acid-base in alanine racemase, aspartate aminotransferase, and poly- l -lysine

Monique Chan-Huot, Alexandra Dos, Reinhard Zander, Shasad Sharif, Peter M. Tolstoy, Shara Compton, Emily Fogle, Michael D. Toney, Ilya Shenderovich, Gleb S. Denisov, Hans Heinrich Limbach

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Abstract

Using 15N solid-state NMR, we have studied protonation and H-bonded states of the cofactor pyridoxal 5′-phosphate (PLP) linked as an internal aldimine in alanine racemase (AlaR), aspartate aminotransferase (AspAT), and poly-l-lysine. Protonation of the pyridine nitrogen of PLP and the coupled proton transfer from the phenolic oxygen (enolimine form) to the aldimine nitrogen (ketoenamine form) is often considered to be a prerequisite to the initial step (transimination) of the enzyme-catalyzed reaction. Indeed, using 15N NMR and H-bond correlations in AspAT, we observe a strong aspartate-pyridine nitrogen H-bond with H located on nitrogen. After hydration, this hydrogen bond is maintained. By contrast, in the case of solid lyophilized AlaR, we find that the pyridine nitrogen is neither protonated nor hydrogen bonded to the proximal arginine side chain. However, hydration establishes a weak hydrogen bond to pyridine. To clarify how AlaR is activated, we performed 13C and 15N solid-state NMR experiments on isotopically labeled PLP aldimines formed by lyophilization with poly-l-lysine. In the dry solid, only the enolimine tautomer is observed. However, a fast reversible proton transfer involving the ketoenamine tautomer is observed after treatment with either gaseous water or gaseous dry HCl. Hydrolysis requires the action of both water and HCl. The formation of an external aldimine with aspartic acid at pH 9 also produces the ketoenamine form stabilized by interaction with a second aspartic acid, probably via a H-bond to the phenolic oxygen. We postulate that O-protonation is an effectual mechanism for the activation of PLP, as is N-protonation, and that enzymes that are incapable of N-protonation employ this mechanism.

Original languageEnglish (US)
Pages (from-to)18160-18175
Number of pages16
JournalJournal of the American Chemical Society
Volume135
Issue number48
DOIs
StatePublished - Dec 4 2013

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Alanine Racemase
Pyridoxal Phosphate
Protonation
Aspartate Aminotransferases
Lysine
Hydrogen
Hydrogen bonds
Phosphates
Nitrogen
Pyridine
Nuclear magnetic resonance
Acids
Aspartic Acid
Proton transfer
Hydration
Protons
Enzymes
Oxygen
Arginine
Freeze Drying

ASJC Scopus subject areas

  • Chemistry(all)
  • Catalysis
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

NMR studies of protonation and hydrogen bond states of internal aldimines of pyridoxal 5′-phosphate acid-base in alanine racemase, aspartate aminotransferase, and poly- l -lysine. / Chan-Huot, Monique; Dos, Alexandra; Zander, Reinhard; Sharif, Shasad; Tolstoy, Peter M.; Compton, Shara; Fogle, Emily; Toney, Michael D.; Shenderovich, Ilya; Denisov, Gleb S.; Limbach, Hans Heinrich.

In: Journal of the American Chemical Society, Vol. 135, No. 48, 04.12.2013, p. 18160-18175.

Research output: Contribution to journalArticle

Chan-Huot, M, Dos, A, Zander, R, Sharif, S, Tolstoy, PM, Compton, S, Fogle, E, Toney, MD, Shenderovich, I, Denisov, GS & Limbach, HH 2013, 'NMR studies of protonation and hydrogen bond states of internal aldimines of pyridoxal 5′-phosphate acid-base in alanine racemase, aspartate aminotransferase, and poly- l -lysine', Journal of the American Chemical Society, vol. 135, no. 48, pp. 18160-18175. https://doi.org/10.1021/ja408988z
Chan-Huot, Monique ; Dos, Alexandra ; Zander, Reinhard ; Sharif, Shasad ; Tolstoy, Peter M. ; Compton, Shara ; Fogle, Emily ; Toney, Michael D. ; Shenderovich, Ilya ; Denisov, Gleb S. ; Limbach, Hans Heinrich. / NMR studies of protonation and hydrogen bond states of internal aldimines of pyridoxal 5′-phosphate acid-base in alanine racemase, aspartate aminotransferase, and poly- l -lysine. In: Journal of the American Chemical Society. 2013 ; Vol. 135, No. 48. pp. 18160-18175.
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abstract = "Using 15N solid-state NMR, we have studied protonation and H-bonded states of the cofactor pyridoxal 5′-phosphate (PLP) linked as an internal aldimine in alanine racemase (AlaR), aspartate aminotransferase (AspAT), and poly-l-lysine. Protonation of the pyridine nitrogen of PLP and the coupled proton transfer from the phenolic oxygen (enolimine form) to the aldimine nitrogen (ketoenamine form) is often considered to be a prerequisite to the initial step (transimination) of the enzyme-catalyzed reaction. Indeed, using 15N NMR and H-bond correlations in AspAT, we observe a strong aspartate-pyridine nitrogen H-bond with H located on nitrogen. After hydration, this hydrogen bond is maintained. By contrast, in the case of solid lyophilized AlaR, we find that the pyridine nitrogen is neither protonated nor hydrogen bonded to the proximal arginine side chain. However, hydration establishes a weak hydrogen bond to pyridine. To clarify how AlaR is activated, we performed 13C and 15N solid-state NMR experiments on isotopically labeled PLP aldimines formed by lyophilization with poly-l-lysine. In the dry solid, only the enolimine tautomer is observed. However, a fast reversible proton transfer involving the ketoenamine tautomer is observed after treatment with either gaseous water or gaseous dry HCl. Hydrolysis requires the action of both water and HCl. The formation of an external aldimine with aspartic acid at pH 9 also produces the ketoenamine form stabilized by interaction with a second aspartic acid, probably via a H-bond to the phenolic oxygen. We postulate that O-protonation is an effectual mechanism for the activation of PLP, as is N-protonation, and that enzymes that are incapable of N-protonation employ this mechanism.",
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T1 - NMR studies of protonation and hydrogen bond states of internal aldimines of pyridoxal 5′-phosphate acid-base in alanine racemase, aspartate aminotransferase, and poly- l -lysine

AU - Chan-Huot, Monique

AU - Dos, Alexandra

AU - Zander, Reinhard

AU - Sharif, Shasad

AU - Tolstoy, Peter M.

AU - Compton, Shara

AU - Fogle, Emily

AU - Toney, Michael D.

AU - Shenderovich, Ilya

AU - Denisov, Gleb S.

AU - Limbach, Hans Heinrich

PY - 2013/12/4

Y1 - 2013/12/4

N2 - Using 15N solid-state NMR, we have studied protonation and H-bonded states of the cofactor pyridoxal 5′-phosphate (PLP) linked as an internal aldimine in alanine racemase (AlaR), aspartate aminotransferase (AspAT), and poly-l-lysine. Protonation of the pyridine nitrogen of PLP and the coupled proton transfer from the phenolic oxygen (enolimine form) to the aldimine nitrogen (ketoenamine form) is often considered to be a prerequisite to the initial step (transimination) of the enzyme-catalyzed reaction. Indeed, using 15N NMR and H-bond correlations in AspAT, we observe a strong aspartate-pyridine nitrogen H-bond with H located on nitrogen. After hydration, this hydrogen bond is maintained. By contrast, in the case of solid lyophilized AlaR, we find that the pyridine nitrogen is neither protonated nor hydrogen bonded to the proximal arginine side chain. However, hydration establishes a weak hydrogen bond to pyridine. To clarify how AlaR is activated, we performed 13C and 15N solid-state NMR experiments on isotopically labeled PLP aldimines formed by lyophilization with poly-l-lysine. In the dry solid, only the enolimine tautomer is observed. However, a fast reversible proton transfer involving the ketoenamine tautomer is observed after treatment with either gaseous water or gaseous dry HCl. Hydrolysis requires the action of both water and HCl. The formation of an external aldimine with aspartic acid at pH 9 also produces the ketoenamine form stabilized by interaction with a second aspartic acid, probably via a H-bond to the phenolic oxygen. We postulate that O-protonation is an effectual mechanism for the activation of PLP, as is N-protonation, and that enzymes that are incapable of N-protonation employ this mechanism.

AB - Using 15N solid-state NMR, we have studied protonation and H-bonded states of the cofactor pyridoxal 5′-phosphate (PLP) linked as an internal aldimine in alanine racemase (AlaR), aspartate aminotransferase (AspAT), and poly-l-lysine. Protonation of the pyridine nitrogen of PLP and the coupled proton transfer from the phenolic oxygen (enolimine form) to the aldimine nitrogen (ketoenamine form) is often considered to be a prerequisite to the initial step (transimination) of the enzyme-catalyzed reaction. Indeed, using 15N NMR and H-bond correlations in AspAT, we observe a strong aspartate-pyridine nitrogen H-bond with H located on nitrogen. After hydration, this hydrogen bond is maintained. By contrast, in the case of solid lyophilized AlaR, we find that the pyridine nitrogen is neither protonated nor hydrogen bonded to the proximal arginine side chain. However, hydration establishes a weak hydrogen bond to pyridine. To clarify how AlaR is activated, we performed 13C and 15N solid-state NMR experiments on isotopically labeled PLP aldimines formed by lyophilization with poly-l-lysine. In the dry solid, only the enolimine tautomer is observed. However, a fast reversible proton transfer involving the ketoenamine tautomer is observed after treatment with either gaseous water or gaseous dry HCl. Hydrolysis requires the action of both water and HCl. The formation of an external aldimine with aspartic acid at pH 9 also produces the ketoenamine form stabilized by interaction with a second aspartic acid, probably via a H-bond to the phenolic oxygen. We postulate that O-protonation is an effectual mechanism for the activation of PLP, as is N-protonation, and that enzymes that are incapable of N-protonation employ this mechanism.

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