NMR studies of the stability, protonation states, and tautomerism of 13C- and 15N-labeled aldimines of the coenzyme pyridoxal 5′-phosphate in water

Monique Chan-Huot, Shasad Sharif, Peter M. Tolstoy, Michael D. Toney, Hans Heinrich Limbach

Research output: Contribution to journalArticle

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Abstract

We have measured the pH-dependent 1H, 13C, and 15N NMR spectra of pyridoxal 5′-phosphate (13C 2-PLP) mixed with equal amounts of either doubly 15N- labeled diaminopropane, 15Nα-labeled l-lysine, or 15Nα-labeled l-lysine as model systems for various intermediates of the transimination reaction in PLP-dependent enzymes. At low pH, only the hydrate and aldehyde forms of PLP and the free protonated diamines are present. Above pH 4, the formation of single- and double-headed aldimines (Schiff bases) with the added diamines is observed, and their 13C and 15N NMR parameters have been characterized. For 1:1 mixtures the single-headed aldimines dominate. In a similar way, the NMR parameters of the geminal diamine formed with diaminopropane at high pH are measured. However, no geminal diamine is formed with l-lysine. In contrast to the aldimine formed with the α-amino group of lysine, the aldimine formed with the α-amino group is unstable at moderately high pH but dominates slightly below pH 10. By analyzing the NMR data, both the mole fractions of the different PLP species and up to 6 different protonation states including their pKa values were obtained. Furthermore, the data show that all Schiff bases are subject to a proton tautomerism along the intramolecular OHN hydrogen bond, where the zwitterionic form is favored before deprotonation occurs at high pH. This observation, as well as the observation that around pH 7 the different PLP species are present in comparable amounts, sheds new light on the mechanism of the transimination reaction.

Original languageEnglish (US)
Pages (from-to)10818-10830
Number of pages13
JournalBiochemistry
Volume49
Issue number51
DOIs
StatePublished - Dec 28 2010

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Pyridoxal Phosphate
Diamines
Protonation
Coenzymes
Lysine
Nuclear magnetic resonance
Schiff Bases
Water
Deprotonation
Hydrates
Aldehydes
Protons
Hydrogen bonds
Enzymes
Hydrogen

ASJC Scopus subject areas

  • Biochemistry

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NMR studies of the stability, protonation states, and tautomerism of 13C- and 15N-labeled aldimines of the coenzyme pyridoxal 5′-phosphate in water. / Chan-Huot, Monique; Sharif, Shasad; Tolstoy, Peter M.; Toney, Michael D.; Limbach, Hans Heinrich.

In: Biochemistry, Vol. 49, No. 51, 28.12.2010, p. 10818-10830.

Research output: Contribution to journalArticle

Chan-Huot, Monique ; Sharif, Shasad ; Tolstoy, Peter M. ; Toney, Michael D. ; Limbach, Hans Heinrich. / NMR studies of the stability, protonation states, and tautomerism of 13C- and 15N-labeled aldimines of the coenzyme pyridoxal 5′-phosphate in water. In: Biochemistry. 2010 ; Vol. 49, No. 51. pp. 10818-10830.
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abstract = "We have measured the pH-dependent 1H, 13C, and 15N NMR spectra of pyridoxal 5′-phosphate (13C 2-PLP) mixed with equal amounts of either doubly 15N- labeled diaminopropane, 15Nα-labeled l-lysine, or 15Nα-labeled l-lysine as model systems for various intermediates of the transimination reaction in PLP-dependent enzymes. At low pH, only the hydrate and aldehyde forms of PLP and the free protonated diamines are present. Above pH 4, the formation of single- and double-headed aldimines (Schiff bases) with the added diamines is observed, and their 13C and 15N NMR parameters have been characterized. For 1:1 mixtures the single-headed aldimines dominate. In a similar way, the NMR parameters of the geminal diamine formed with diaminopropane at high pH are measured. However, no geminal diamine is formed with l-lysine. In contrast to the aldimine formed with the α-amino group of lysine, the aldimine formed with the α-amino group is unstable at moderately high pH but dominates slightly below pH 10. By analyzing the NMR data, both the mole fractions of the different PLP species and up to 6 different protonation states including their pKa values were obtained. Furthermore, the data show that all Schiff bases are subject to a proton tautomerism along the intramolecular OHN hydrogen bond, where the zwitterionic form is favored before deprotonation occurs at high pH. This observation, as well as the observation that around pH 7 the different PLP species are present in comparable amounts, sheds new light on the mechanism of the transimination reaction.",
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T1 - NMR studies of the stability, protonation states, and tautomerism of 13C- and 15N-labeled aldimines of the coenzyme pyridoxal 5′-phosphate in water

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AU - Sharif, Shasad

AU - Tolstoy, Peter M.

AU - Toney, Michael D.

AU - Limbach, Hans Heinrich

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N2 - We have measured the pH-dependent 1H, 13C, and 15N NMR spectra of pyridoxal 5′-phosphate (13C 2-PLP) mixed with equal amounts of either doubly 15N- labeled diaminopropane, 15Nα-labeled l-lysine, or 15Nα-labeled l-lysine as model systems for various intermediates of the transimination reaction in PLP-dependent enzymes. At low pH, only the hydrate and aldehyde forms of PLP and the free protonated diamines are present. Above pH 4, the formation of single- and double-headed aldimines (Schiff bases) with the added diamines is observed, and their 13C and 15N NMR parameters have been characterized. For 1:1 mixtures the single-headed aldimines dominate. In a similar way, the NMR parameters of the geminal diamine formed with diaminopropane at high pH are measured. However, no geminal diamine is formed with l-lysine. In contrast to the aldimine formed with the α-amino group of lysine, the aldimine formed with the α-amino group is unstable at moderately high pH but dominates slightly below pH 10. By analyzing the NMR data, both the mole fractions of the different PLP species and up to 6 different protonation states including their pKa values were obtained. Furthermore, the data show that all Schiff bases are subject to a proton tautomerism along the intramolecular OHN hydrogen bond, where the zwitterionic form is favored before deprotonation occurs at high pH. This observation, as well as the observation that around pH 7 the different PLP species are present in comparable amounts, sheds new light on the mechanism of the transimination reaction.

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