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Solvent isotope-induced equilibrium perturbation for isocitrate lyase
Christine E Quartararo1, Timin Hadi, Sean M Cahill
1Department of Biochemistry, Albert Einstein College of Medicine , 1300 Morris Park Avenue, Bronx, New York 10461, United States.
Biochemistry
|November 23, 2013
Summary
Isocitrate lyase (ICL) enzyme activity was studied using deuterium. Researchers observed transient changes in reaction equilibrium and isotopic composition of molecules, particularly succinate and isocitrate.
Area of Science:
- Biochemistry
- Enzymology
- Metabolic pathways
Background:
- Isocitrate lyase (ICL) is crucial for Mycobacterium tuberculosis persistence.
- ICL initiates the glyoxylate shunt, enabling anaplerosis of citric acid cycle intermediates.
- Nutrient limitation triggers the glyoxylate shunt's importance.
Purpose of the Study:
- Investigate the mechanism of isocitrate cleavage by ICL.
- Quantify solvent kinetic isotope effects (KIEs) in the reaction.
- Monitor isotopic composition changes during the reaction in D₂O.
Main Methods:
- Utilized solvent kinetic isotope effects (KIEs) with D₂O.
- Employed nuclear magnetic resonance (NMR) spectroscopy for monitoring.
- Applied mass spectrometry to track isotopic species concentrations.
Main Results:
- Observed significant KIEs for isocitrate cleavage.
- Detected transient equilibrium perturbations in D₂O.
- Identified dideuterated succinate, protiated glyoxylate, and monodeuterated isocitrate at equilibrium.
Conclusions:
- Solvent KIEs indicate a proton transfer step in the ICL reaction mechanism.
- The glyoxylate shunt equilibrium is perturbed by solvent deuterium.
- NMR and MS confirmed dynamic changes in isotopic composition during the reaction.
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