Related Experiment Video
Updated: Aug 8, 2026
08:59
Use of a Multi-compartment Dynamic Single Enzyme Phantom for Studies of Hyperpolarized Magnetic Resonance Agents
Published on: April 15, 2016
31P magnetization transfer in the phosphoglyceromutase-enolase coupled enzyme system
B E Chapman1, I M Stewart, B T Bulliman
1Department of Biochemistry, University of Sydney, NSW, Australia.
European Biophysics Journal : EBJ
|January 1, 1988
Summary
Phosphoryl group exchange rates were measured for key glycolytic intermediates using 31P NMR spectroscopy. Two-dimensional NMR offers a robust method for analyzing complex enzyme systems like phosphoglyceromutase-enolase.
Area of Science:
- Biochemistry
- Enzymology
- Spectroscopy
Background:
- Phosphoryl group transfer is central to cellular energy metabolism.
- The phosphoglyceromutase-enolase enzyme system catalyzes key steps in glycolysis.
- Accurate measurement of exchange rates is crucial for understanding enzyme mechanisms.
Purpose of the Study:
- To quantify the phosphoryl group exchange rates between 2-phosphoglycerate, 3-phosphoglycerate, and phosphoenolpyruvate.
- To evaluate the utility of one- and two-dimensional 31P NMR spectroscopy for studying enzyme-catalyzed exchange reactions.
- To compare the effectiveness of saturation transfer and two-dimensional exchange NMR methods.
Main Methods:
- Utilized one- and two-dimensional 31P NMR spectroscopy.
- Employed saturation transfer with selective irradiation (DANTE pulse sequence) for one-dimensional experiments.
- Applied a two-dimensional magnetization exchange experiment with back transformation analysis.
Main Results:
- Successfully measured phosphoryl group exchange rates within the phosphoglyceromutase-enolase system.
- Demonstrated the capability of saturation transfer NMR for analyzing three-site exchange.
- Showcased the advantage of two-dimensional NMR in avoiding selective saturation challenges.
- Obtained exchange rate constants from 2D NMR in good agreement with 1D saturation transfer results.
Conclusions:
- Two-dimensional 31P NMR provides a reliable method for determining exchange rates in complex enzymatic systems.
- The phosphoglyceromutase-enolase system's phosphoryl exchange dynamics were quantitatively characterized.
- This study validates advanced NMR techniques for mechanistic enzyme studies.
Related Concept Videos
01:19Enzyme Kinetics
Enzymes speed up reactions by lowering the activation energy of the reactants. The speed at which the enzyme turns reactants into products is called the rate of reaction. Several factors impact the rate of reaction, including the number of available reactants. Enzyme kinetics is the study of how an enzyme changes the rate of a reaction.
Scientists typically study enzyme kinetics with a fixed amount of enzyme in the controlled environment of a test tube. When more reactant, or substrate, is...
Scientists typically study enzyme kinetics with a fixed amount of enzyme in the controlled environment of a test tube. When more reactant, or substrate, is...
01:58Cooperative Allosteric Transitions
Cooperative allosteric transitions can occur in multimeric proteins, where each subunit of the protein has its own ligand-binding site. When a ligand binds to any of these subunits, it triggers a conformational change that affects the binding sites in the other subunits; this can change the affinity of the other sites for their respective ligands. The ability of the protein to change the shape of its binding site is attributed to the presence of a mix of flexible and stable segments in the...
01:58Cooperative Allosteric Transitions
Cooperative allosteric transitions can occur in multimeric proteins, where each subunit of the protein has its own ligand-binding site. When a ligand binds to any of these subunits, it triggers a conformational change that affects the binding sites in the other subunits; this can change the affinity of the other sites for their respective ligands. The ability of the protein to change the shape of its binding site is attributed to the presence of a mix of flexible and stable segments in the...
01:58Cooperative Allosteric Transitions
Cooperative allosteric transitions can occur in multimeric proteins, where each subunit of the protein has its own ligand-binding site. When a ligand binds to any of these subunits, it triggers a conformational change that affects the binding sites in the other subunits; this can change the affinity of the other sites for their respective ligands. The ability of the protein to change the shape of its binding site is attributed to the presence of a mix of flexible and stable segments in the...
01:15Insensitive Nuclei Enhanced by Polarization Transfer (INEPT)
Insensitive Nuclei Enhanced by Polarization Transfer (INEPT) is an advanced Nuclear Magnetic Resonance (NMR) technique specifically designed to detect and enhance the signals of low-abundance nuclei, such as carbon-13 and nitrogen-15, in small molecules. The fundamental principle behind INEPT is the transfer of polarization from a more abundant and highly polarizable nucleus, typically hydrogen-1, to the low-abundance nucleus of interest. This process effectively boosts the NMR signal of the...