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Turnover Number and Catalytic Efficiency01:19

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The turnover number of an enzyme is the maximum number of substrate molecules it can transform per unit time. Turnover numbers for most enzymes range from 1 to 1000 molecules per second. Catalase has the known highest turnover number, capable of converting up to 2.8×106 molecules of hydrogen peroxide into water and oxygen per second. Lysozyme has the lowest known turnover number of half a molecule per second.
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Acetoacetic ester synthesis is a method to obtain ketones from alkyl halides and β-keto esters. The reaction occurs in the presence of an alkoxide base that abstracts the acidic proton of the β-keto esters. The step results in an enolate ion which is doubly stabilized. The enolate then reacts with an alkyl halide via the SN2 process to produce an alkylated ester intermediate with a new C–C bond. The hydrolysis of the intermediate, followed by acidification, results in an...
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The pinacol and McMurry reactions involve the reductive coupling of ketones or aldehydes. Similarly, the bimolecular reductive coupling of two ester molecules in the presence of sodium metal in an aprotic solvent yields an α-hydroxy ketone product. The α-hydroxy ketone is also called acyloin, so the reaction is referred to as ‘acyloin condensation.’
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By replacing an α-hydrogen with a halogen, acid-catalyzed α-halogenation of aldehydes or ketones yields a monohalogenated product
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Substrate Turnover Dynamics Guide Ketol-Acid Reductoisomerase Redesign for Increased Specific Activity.

Elijah Karvelis1,2, Chloe Swanson1,2, Bruce Tidor1,2,3

  • 1Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.

ACS Catalysis
|July 25, 2024
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Summary
This summary is machine-generated.

Researchers engineered ketol-acid reductoisomerase (KARI) for better isobutanol production by analyzing enzyme dynamics. This approach identified mutations significantly boosting catalytic activity, offering a new strategy for enzyme engineering.

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Area of Science:

  • Biocatalysis
  • Enzyme Engineering
  • Computational Chemistry

Background:

  • Enzyme adaptation for industrial applications is limited by incomplete control over catalytic functions.
  • Ketol-acid reductoisomerase (KARI) is crucial for industrial isobutanol production.
  • Traditional enzyme redesign focuses on ground and transition states, potentially missing dynamic effects.

Purpose of the Study:

  • To develop a rational computational approach to enhance enzyme catalytic activity.
  • To identify mutations in KARI that increase its specific activity for isobutanol production.
  • To investigate the role of enzyme dynamics in catalysis.

Main Methods:

  • Utilized path sampling techniques and QM/MM simulations to model KARI substrate turnover dynamics.
  • Employed machine learning to identify conformational features correlated with productive catalysis.
  • Applied multistate protein redesign to select mutations stabilizing reactive conformations.

Main Results:

  • Identified specific enzyme-substrate complex conformations promoting catalysis.
  • Generated eight enzyme mutants with significantly improved calculated specific activity.
  • Observed up to a (2 ± 1) × 10^4-fold increase in calculated kcat for the fastest variant.

Conclusions:

  • Analyzing complete enzyme turnover dynamics provides insights for enzyme engineering.
  • Stabilizing reaction-promoting conformations is an effective strategy for enhancing enzyme catalysts.
  • This rational approach advances the design of enzymes for industrial applications like isobutanol synthesis.