Related Experiment Video
Updated: Jul 15, 2026
06:18
Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue
Published on: June 21, 2018
Lactate dehydrogenase undergoes a substantial structural change to bind its substrate.
Linlin Qiu1, Miriam Gulotta, Robert Callender
1Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY, USA.
Biophysical Journal
|May 8, 2007
Summary
Lactate dehydrogenase (LDH) uses a two-step process to form a ternary binding intermediate, involving distinct submillisecond kinetics. Protein stabilizers and destabilizers modulate this process, suggesting conformational flexibility in LDH/NADH binding.
Area of Science:
- Biochemistry
- Enzyme kinetics
- Protein dynamics
Background:
- Lactate dehydrogenase (LDH) is crucial for cellular metabolism.
- Understanding enzyme-substrate interactions is key to drug development.
- Early binding events can significantly impact overall enzyme function.
Purpose of the Study:
- To investigate the kinetics and thermodynamics of early ternary complex formation in LDH.
- To characterize the 'encounter complex' intermediate.
- To explore the influence of protein stabilizers and destabilizers on binding kinetics.
Main Methods:
- Temperature-jump relaxation spectroscopy was employed.
- Kinetics were measured as a function of temperature.
- The effects of urea (destabilizer) and TMAO (stabilizer) were assessed.
Main Results:
- Two distinct submillisecond processes were identified in encounter complex formation.
- Binding kinetics exhibited inverse Arrhenius behavior and a temperature-dependent enthalpy.
- TMAO slowed the reaction, while urea accelerated it.
- Results support a 'select fit' model with LDH/NADH existing in equilibrium between open and closed conformations.
Conclusions:
- LDH/NADH exists in a dynamic equilibrium of conformations, with a minority 'open' state being competent for rapid substrate binding.
- The binding-competent state likely has an exposed active site, facilitating diffusion-limited ligand association.
- Thermodynamic data suggest differences in hydrogen bonding and solvation between conformational states.
More Related Videos
Related Concept Videos
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...
02:40Ligand Binding Sites
Proteins are dynamic macromolecules that carry out a wide variety of essential processes; however, the activities of most proteins depend on their interactions with other molecules or ions, known as ligands.
Protein-ligand interactions are quite specific; even though numerous potential ligands surround a cellular protein at any given time, only a particular ligand can bind to that protein. Moreover, a ligand binds only to a dedicated area on the surface of the protein, known as the...
Protein-ligand interactions are quite specific; even though numerous potential ligands surround a cellular protein at any given time, only a particular ligand can bind to that protein. Moreover, a ligand binds only to a dedicated area on the surface of the protein, known as the...
00:49Ligand Binding and Linkage
Allosteric proteins have more than one ligand binding site; the binding of a ligand to any of these sites influences the binding of ligands to the other sites. When a protein is allosteric, its binding sites are called coupled or linked. In the case of enzymes, the site that binds to the substrate is known as the active site and the other site is known as the regulatory site. When a ligand binds to the regulatory site, this leads to conformational changes in the protein that can influence the...
01:15Pyruvate Oxidation
After glycolysis, the charged pyruvate molecules enter the mitochondria via active transport and undergo three enzymatic reactions. These reactions ensure that pyruvate can enter the next metabolic pathway so that energy stored in the pyruvate molecules can be harnessed by the cells.
First, the enzyme pyruvate dehydrogenase removes the carboxyl group from pyruvate and releases it as carbon dioxide. The stripped molecule is then oxidized and releases electrons, which are then picked up by NAD+...
First, the enzyme pyruvate dehydrogenase removes the carboxyl group from pyruvate and releases it as carbon dioxide. The stripped molecule is then oxidized and releases electrons, which are then picked up by NAD+...