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Related Concept Videos

Allosteric Proteins-ATCase01:19

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Binding sites linkages can regulate a protein's function.  For example, enzyme activity is often regulated through a feedback mechanism where the end product of the biochemical process serves as an inhibitor.
Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate to  N-carbamoyl-L-aspartate. This reaction is the first step in pyrimidine biosynthesis. UTP and CTP, the end products of the pyrimidine synthesis...
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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...
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tRNA Activation

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Aminoacyl-tRNA synthetases are present in both eukaryotes and bacteria. Though eukaryotes have 20 different aminoacyl-tRNA synthetases to couple to 20 amino acids, many bacteria do not have genes for all of these aminoacyl-tRNA synthetases. Despite this, they still use all 20 amino acids to synthesize their proteins. For instance, some bacteria do not have the gene encoding the enzyme that couples glutamine with its partner tRNA. In these organisms, one enzyme adds glutamic acid to all of the...
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Cooperative Allosteric Transitions01:58

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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...
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Allosteric Regulation01:08

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Allosteric regulation of enzymes occurs when the binding of an effector molecule to a site that is different from the active site causes a change in the enzymatic activity. This alternate site is called an allosteric site, and an enzyme can contain more than one of these sites. Allosteric regulation can either be positive or negative, resulting in an increase or decrease in enzyme activity. Most enzymes that display allosteric regulation are metabolic enzymes involved in the degradation or...
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Related Experiment Video

Updated: Aug 26, 2025

PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase
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PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase

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Dynamic allostery in substrate binding by human thymidylate synthase.

Jeffrey P Bonin1, Paul J Sapienza2, Andrew L Lee1,2

  • 1Department of Biochemistry and Biophysics, School of Medicine, University of North Carolina, Chapel Hill, United States.

Elife
|October 6, 2022
PubMed
Summary
This summary is machine-generated.

Human thymidylate synthase (hTS) cooperativity arises from enzyme rigidification upon substrate binding, not conformational selection. This dynamic allostery involves the flexible N-terminus, offering novel insights into cancer enzyme regulation.

Keywords:
NMRallosteryconformational entropycooperativityhumanmolecular biophysicsprotein dynamicsstructural biologythymidylate synthase

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Last Updated: Aug 26, 2025

PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase
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Area of Science:

  • Biochemistry
  • Enzymology
  • Structural Biology

Background:

  • Human thymidylate synthase (hTS) is a crucial enzyme for DNA replication and a key target in cancer therapy.
  • Understanding the regulatory mechanisms of hTS, particularly its substrate binding cooperativity, is vital for developing effective anti-cancer drugs.

Purpose of the Study:

  • To elucidate the molecular mechanism underlying the binding cooperativity of the nucleotide substrate to human thymidylate synthase.
  • To investigate the role of conformational changes and dynamics in hTS regulation and allostery.

Main Methods:

  • Utilized methyl-based Carr-Purcell-Meiboom-Gill (CPMG) and Chemical Exchange Saturation Transfer (CEST) Nuclear Magnetic Resonance (NMR) spectroscopy.
  • Determined methyl rotation axis order parameters using 2H transverse relaxation rates to assess enzyme dynamics.

Main Results:

  • Identified residues exhibiting 3-state exchange and concerted conformational switching between active and inactive states in the apo enzyme.
  • Demonstrated that enzyme rigidification upon substrate binding, rather than conformational selection, drives entropically-driven cooperativity.
  • Showcased that N-terminal truncation and product binding abolish this rigidification and cooperativity, highlighting the role of the N-terminus in dynamic allostery.

Conclusions:

  • Substrate binding cooperativity in hTS is primarily mediated by enzyme rigidification, a novel mechanism of dynamic allostery involving the flexible N-terminus.
  • These findings provide a deep understanding of hTS regulation, crucial for its targeting in cancer therapy.