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Related Concept Videos

Allosteric Proteins-ATCase01:19

Allosteric Proteins-ATCase

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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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Cyclohexane does not exist in a planar form due to the high angle and torsional strain it would experience in the planar structure. Instead, it adopts non-planar chair and boat conformations.
The chair form is the most stable and derives its name from its resemblance to the “easy chair.” In the chair conformation, two carbon atoms are arranged out-of-plane — one above and one below, minimizing the torsional strain. In the chair form, the bond angle is very close to the ideal...
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Cooperative Allosteric Transitions01:58

Cooperative Allosteric Transitions

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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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Ligand Binding and Linkage00:49

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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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Chair Conformation of Cyclohexane02:02

Chair Conformation of Cyclohexane

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The chair conformation is the most stable form of cyclohexane due to the absence of angle and torsional strain. The absence of angle strain is a result of cyclohexane’s bond angle being very close to the ideal tetrahedral bond angle of 109.5° in its chair conformer. Similarly, the torsional strain is also absent owing to the perfectly staggered arrangement of bonds.
The hydrogen atoms linked to carbons are arranged in two different axial and equatorial orientations to achieve this...
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¹H NMR of Conformationally Flexible Molecules: Temporal Resolution00:52

¹H NMR of Conformationally Flexible Molecules: Temporal Resolution

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At room temperature, the chair conformer of cyclohexane undergoes rapid ring flipping between two equivalent chair conformers at a rate of approximately 105 times per second. These two chair conformers are in equilibrium. The rapid ring flipping results in the interconversion of the axial proton to an equatorial proton and an equatorial to the axial proton. Such interconversions are too rapid and cannot be detected on the NMR timescale. Hence, the NMR spectrometer cannot distinguish between the...
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Conformational heterogeneity suggests multiple substrate binding modes in CYP106A2.

Nathan R Wong1, Reethy Sundar1, Sophia Kazanis2

  • 1Dept. of Biochemistry, Brandeis University, 415 South St., Waltham, MA 02454, United States of America.

Journal of Inorganic Biochemistry
|February 2, 2023
PubMed
Summary

This study details the structural analysis of cytochrome P450meg (CYP106A2) using NMR. Findings reveal that substrate binding does not reduce enzyme structural heterogeneity, suggesting multiple binding modes.

Keywords:
Cytochrome P450Enzyme conformationsNuclear magnetic resonanceParamagnetismRegiochemistryStereochemistrySubstrate

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Area of Science:

  • Biochemistry
  • Enzymology
  • Structural Biology

Background:

  • Cytochrome P450meg (CYP106A2) is a bacterial enzyme known for hydroxylating diverse substrates, including steroids.
  • Previous studies indicate variable regio- and stereochemistry in CYP106A2 hydroxylation, with potential for multiple hydroxylation sites and lack of stereospecificity.

Purpose of the Study:

  • To provide comprehensive backbone resonance assignments for CYP106A2 using multidimensional NMR.
  • To investigate the structural impact of substrate binding on CYP106A2.
  • To explore substrate binding modes using paramagnetic relaxation enhancement (PRE).

Main Methods:

  • Multidimensional nuclear magnetic resonance (NMR) experiments with uniformly and selectively isotopically labeled CYP106A2.
  • Analysis of resonance broadening and splitting upon substrate binding.
  • Paramagnetic relaxation enhancement (PRE) measurements with three different substrates.

Main Results:

  • Complete backbone 15N, 1H, and 13C resonance assignments for CYP106A2 were established.
  • Substrate binding did not reduce structural heterogeneity, unlike in other P450 enzymes (CYP101A1, MycG).
  • PRE data suggest that multiple substrate binding modes are utilized at saturating concentrations.

Conclusions:

  • CYP106A2 maintains structural heterogeneity even with substrate bound.
  • The enzyme likely employs multiple substrate binding orientations, contributing to its broad substrate specificity and hydroxylation patterns.