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Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry
Published on: September 13, 2014
Cofactor binding to Escherichia coli D-3-phosphoglycerate dehydrogenase induces multiple conformations which alter
Gregory A Grant1, Zhiqin Hu, Xiao Lan Xu
1Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110, USA. ggrant@pcg.wustl.edu
The Journal of Biological Chemistry
|August 17, 2002
Summary
NADH binding to Escherichia coli d-3-phosphoglycerate dehydrogenase alters inhibitor binding cooperativity. This cofactor-induced conformational change affects enzyme inhibition, impacting catalytic activity.
Area of Science:
- Biochemistry
- Enzyme kinetics
- Allosteric regulation
Background:
- Escherichia coli d-3-phosphoglycerate dehydrogenase is inhibited by L-serine.
- Phosphate was previously shown to affect inhibitor binding cooperativity, but the mechanism was unclear.
Purpose of the Study:
- To investigate the role of intrinsically bound NADH in modulating L-serine inhibition of Escherichia coli d-3-phosphoglycerate dehydrogenase.
- To elucidate the mechanism by which NADH affects enzyme cooperativity and inhibitor binding.
Main Methods:
- Equilibrium binding experiments were performed to study the interaction of L-serine and NADH with the enzyme.
- The effect of NADH on inhibitor binding cooperativity was compared to NAD+ and alpha-ketoglutarate.
Main Results:
- Intrinsically bound NADH significantly alters L-serine binding cooperativity, reducing both positive and negative cooperativity.
- Evidence suggests negative cooperativity in NADH binding and the existence of at least two NADH-induced enzyme conformations.
- NADH binding increases affinity for the first inhibitor ligand and alters cooperativity, an effect specific to NADH.
Conclusions:
- Cofactor-bound NADH plays a crucial role in regulating the allosteric inhibition of d-3-phosphoglycerate dehydrogenase by L-serine.
- NADH-induced conformational changes in the enzyme lead to differential inhibition of catalytic activity.
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