Solvent isotope-induced equilibrium perturbation for isocitrate lyase

Christine E. Quartararo, Timin Hadi, Sean M. Cahill, John S. Blanchard

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Isocitrate lyase (ICL) catalyzes the reversible retro-aldol cleavage of isocitrate to generate glyoxylate and succinate. ICL is the first enzyme of the glyoxylate shunt, which allows for the anaplerosis of citric acid cycle intermediates under nutrient limiting conditions. In Mycobacterium tuberculosis, the source of ICL for these studies, ICL is vital for the persistence phase of the bacterium's life cycle. Solvent kinetic isotope effects (KIEs) in the direction of isocitrate cleavage (D2OV = 2.0 ± 0.1, and D 2O[V/Kisocitrate] = 2.2 ± 0.3) arise from the initial deprotonation of the C2 hydroxyl group of isocitrate or the protonation of the aci-acid of the succinate product of the isocitrate aldol cleavage by a solvent-derived proton. This KIE suggested that an equilibrium mixture of all protiated isocitrate, glyoxylate, and succinate prepared in D2O would undergo transient changes in equilibrium concentrations as a result of the solvent KIE and solvent-derived deuterium incorporation into both succinate and isocitrate. No change in the isotopic composition of glyoxylate was expected or observed. We have directly monitored the changing concentrations of all isotopic species of all reactants and products using a combination of nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. Continuous monitoring of glyoxylate by 1H NMR spectroscopy shows a clear equilibrium perturbation in D2O. The final equilibrium isotopic composition of reactants in D2O revealed dideuterated succinate, protiated glyoxylate, and monodeuterated isocitrate, with the transient appearance and disappearance of monodeuterated succinate. A model for the equilibrium perturbation of substrate species and their time-dependent isotopic composition is presented.

Original languageEnglish (US)
Pages (from-to)9286-9293
Number of pages8
JournalBiochemistry
Volume52
Issue number51
DOIs
StatePublished - Dec 23 2013

Fingerprint

Isocitrate Lyase
Isotopes
Succinic Acid
Nuclear magnetic resonance spectroscopy
Kinetics
Magnetic Resonance Spectroscopy
Chemical analysis
Deprotonation
Citric Acid Cycle
Deuterium
Protonation
isocitric acid
Life Cycle Stages
Mycobacterium tuberculosis
Hydroxyl Radical
Nutrients
Mass spectrometry
glyoxylic acid
Protons
Life cycle

ASJC Scopus subject areas

  • Biochemistry
  • Medicine(all)

Cite this

Solvent isotope-induced equilibrium perturbation for isocitrate lyase. / Quartararo, Christine E.; Hadi, Timin; Cahill, Sean M.; Blanchard, John S.

In: Biochemistry, Vol. 52, No. 51, 23.12.2013, p. 9286-9293.

Research output: Contribution to journalArticle

Quartararo, Christine E. ; Hadi, Timin ; Cahill, Sean M. ; Blanchard, John S. / Solvent isotope-induced equilibrium perturbation for isocitrate lyase. In: Biochemistry. 2013 ; Vol. 52, No. 51. pp. 9286-9293.
@article{ba964d63462446728838a9f09d21682d,
title = "Solvent isotope-induced equilibrium perturbation for isocitrate lyase",
abstract = "Isocitrate lyase (ICL) catalyzes the reversible retro-aldol cleavage of isocitrate to generate glyoxylate and succinate. ICL is the first enzyme of the glyoxylate shunt, which allows for the anaplerosis of citric acid cycle intermediates under nutrient limiting conditions. In Mycobacterium tuberculosis, the source of ICL for these studies, ICL is vital for the persistence phase of the bacterium's life cycle. Solvent kinetic isotope effects (KIEs) in the direction of isocitrate cleavage (D2OV = 2.0 ± 0.1, and D 2O[V/Kisocitrate] = 2.2 ± 0.3) arise from the initial deprotonation of the C2 hydroxyl group of isocitrate or the protonation of the aci-acid of the succinate product of the isocitrate aldol cleavage by a solvent-derived proton. This KIE suggested that an equilibrium mixture of all protiated isocitrate, glyoxylate, and succinate prepared in D2O would undergo transient changes in equilibrium concentrations as a result of the solvent KIE and solvent-derived deuterium incorporation into both succinate and isocitrate. No change in the isotopic composition of glyoxylate was expected or observed. We have directly monitored the changing concentrations of all isotopic species of all reactants and products using a combination of nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. Continuous monitoring of glyoxylate by 1H NMR spectroscopy shows a clear equilibrium perturbation in D2O. The final equilibrium isotopic composition of reactants in D2O revealed dideuterated succinate, protiated glyoxylate, and monodeuterated isocitrate, with the transient appearance and disappearance of monodeuterated succinate. A model for the equilibrium perturbation of substrate species and their time-dependent isotopic composition is presented.",
author = "Quartararo, {Christine E.} and Timin Hadi and Cahill, {Sean M.} and Blanchard, {John S.}",
year = "2013",
month = "12",
day = "23",
doi = "10.1021/bi4013319",
language = "English (US)",
volume = "52",
pages = "9286--9293",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "51",

}

TY - JOUR

T1 - Solvent isotope-induced equilibrium perturbation for isocitrate lyase

AU - Quartararo, Christine E.

AU - Hadi, Timin

AU - Cahill, Sean M.

AU - Blanchard, John S.

PY - 2013/12/23

Y1 - 2013/12/23

N2 - Isocitrate lyase (ICL) catalyzes the reversible retro-aldol cleavage of isocitrate to generate glyoxylate and succinate. ICL is the first enzyme of the glyoxylate shunt, which allows for the anaplerosis of citric acid cycle intermediates under nutrient limiting conditions. In Mycobacterium tuberculosis, the source of ICL for these studies, ICL is vital for the persistence phase of the bacterium's life cycle. Solvent kinetic isotope effects (KIEs) in the direction of isocitrate cleavage (D2OV = 2.0 ± 0.1, and D 2O[V/Kisocitrate] = 2.2 ± 0.3) arise from the initial deprotonation of the C2 hydroxyl group of isocitrate or the protonation of the aci-acid of the succinate product of the isocitrate aldol cleavage by a solvent-derived proton. This KIE suggested that an equilibrium mixture of all protiated isocitrate, glyoxylate, and succinate prepared in D2O would undergo transient changes in equilibrium concentrations as a result of the solvent KIE and solvent-derived deuterium incorporation into both succinate and isocitrate. No change in the isotopic composition of glyoxylate was expected or observed. We have directly monitored the changing concentrations of all isotopic species of all reactants and products using a combination of nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. Continuous monitoring of glyoxylate by 1H NMR spectroscopy shows a clear equilibrium perturbation in D2O. The final equilibrium isotopic composition of reactants in D2O revealed dideuterated succinate, protiated glyoxylate, and monodeuterated isocitrate, with the transient appearance and disappearance of monodeuterated succinate. A model for the equilibrium perturbation of substrate species and their time-dependent isotopic composition is presented.

AB - Isocitrate lyase (ICL) catalyzes the reversible retro-aldol cleavage of isocitrate to generate glyoxylate and succinate. ICL is the first enzyme of the glyoxylate shunt, which allows for the anaplerosis of citric acid cycle intermediates under nutrient limiting conditions. In Mycobacterium tuberculosis, the source of ICL for these studies, ICL is vital for the persistence phase of the bacterium's life cycle. Solvent kinetic isotope effects (KIEs) in the direction of isocitrate cleavage (D2OV = 2.0 ± 0.1, and D 2O[V/Kisocitrate] = 2.2 ± 0.3) arise from the initial deprotonation of the C2 hydroxyl group of isocitrate or the protonation of the aci-acid of the succinate product of the isocitrate aldol cleavage by a solvent-derived proton. This KIE suggested that an equilibrium mixture of all protiated isocitrate, glyoxylate, and succinate prepared in D2O would undergo transient changes in equilibrium concentrations as a result of the solvent KIE and solvent-derived deuterium incorporation into both succinate and isocitrate. No change in the isotopic composition of glyoxylate was expected or observed. We have directly monitored the changing concentrations of all isotopic species of all reactants and products using a combination of nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. Continuous monitoring of glyoxylate by 1H NMR spectroscopy shows a clear equilibrium perturbation in D2O. The final equilibrium isotopic composition of reactants in D2O revealed dideuterated succinate, protiated glyoxylate, and monodeuterated isocitrate, with the transient appearance and disappearance of monodeuterated succinate. A model for the equilibrium perturbation of substrate species and their time-dependent isotopic composition is presented.

UR - http://www.scopus.com/inward/record.url?scp=84892605464&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84892605464&partnerID=8YFLogxK

U2 - 10.1021/bi4013319

DO - 10.1021/bi4013319

M3 - Article

C2 - 24261638

AN - SCOPUS:84892605464

VL - 52

SP - 9286

EP - 9293

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 51

ER -