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Related Concept Videos

C–C Bond Cleavage: Retro-Aldol Reaction00:57

C–C Bond Cleavage: Retro-Aldol Reaction

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The reverse of the aldol addition reaction is called the retro-aldol reaction. Here, the carbon–carbon bond in the aldol product is cleaved under acidic or basic conditions to form two molecules of carbonyl compounds. The mechanism of the reaction consists of three steps.
In the first step, as depicted in Figure 1, the base deprotonates the β-hydroxy ketone at the hydroxyl group to form an alkoxide ion.
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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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As depicted in Figure 1, base-catalyzed aldol addition involves adding two carbonyl compounds in aqueous sodium hydroxide to form a β-hydroxy carbonyl compound.
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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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ATP Synthase: Mechanism01:48

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In animals, the mitochondrial F1F0 ATP synthase is the key protein that synthesizes ATP molecules through a complex catalytic mechanism. While the nuclear genome encodes the majority of ATP synthase subunits, the mitochondrial genome encodes some of the enzyme's most critical components. The formation of this multi-subunit enzyme is a complex multi-step process regulated at the level of transcription, translation, and assembly. Defects in one or more of these steps can result in decreased...
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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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Distal Mutations in a Designed Retro-Aldolase Alter Loop Dynamics to Shift and Accelerate the Rate-Limiting Step.

Serena E Hunt1,2, Cindy Klaus1,2, Aqza E John3

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Distal mutations in enzymes, distant from the active site, enhance catalytic efficiency when combined with active-site mutations. These distal residues are crucial for enzyme design by influencing enzyme structure and dynamics.

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Area of Science:

  • Enzyme catalysis
  • Protein engineering
  • Biochemistry

Background:

  • Amino acid residues distant from an enzyme's active site can influence catalysis, but their mechanisms are unclear.
  • Directed evolution and computational design are powerful tools for enzyme engineering.

Purpose of the Study:

  • Investigate the structural, functional, and mechanistic impacts of distal and active-site mutations in the retro-aldolase RA95.
  • Elucidate the role of distal residues in enzyme catalysis and active site dynamics.

Main Methods:

  • Directed evolution of retro-aldolase RA95.
  • X-ray crystallography and molecular dynamics simulations.
  • Kinetic solvent viscosity effects and electric field calculations.

Main Results:

  • Active-site mutations improved catalytic efficiency 3,600-fold; distal mutations alone showed no improvement.
  • Combined active-site and distal mutations yielded a 6-fold further increase in efficiency (epistasis).
  • Distal mutations promote active site opening by altering loop dynamics, accelerating chemical transformation 100-fold.

Conclusions:

  • Distal residues play a critical role in shaping the enzyme's active site environment.
  • Distal mutations facilitate essential structural dynamics for efficient enzyme catalysis.
  • Findings offer valuable insights for the rational design of enzymes with enhanced catalytic properties.