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Related Concept Videos

Restriction Enzymes01:11

Restriction Enzymes

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Restriction enzymes are bacterial enzymes used to cut DNA in a sequence-specific manner. To cleave DNA, they bind to specific palindromic sequences called restriction sites. Such palindromic DNA sequences or inverted repeats are commonly found in regions of functional significance, such as the origin of replication, gene operator sites, and regions containing transcription termination signals.
The host bacteria protect their own genomic DNA from these enzymes by methylating these sites. Some...
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Enzymes02:34

Enzymes

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Inside living organisms, enzymes act as catalysts for many biochemical reactions involved in cellular metabolism. The role of enzymes is to reduce the activation energies of biochemical reactions by forming complexes with its substrates. The lowering of activation energies favor an increase in the rates of biochemical reactions.
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Introduction to Enzymes01:22

Introduction to Enzymes

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The use of enzymes by humans dates to 7000 BCE. Humans first used enzymes to ferment sugars and produce alcohol without knowing that this was an enzyme-catalyzed reaction. Wilhelm Kuhne coined the term 'enzyme' in 1877 from the Greek words ‘en’ meaning ‘in’ or ‘within’ and ‘zyme’ meaning ‘yeast.’
Most enzymes are proteins that speed up biochemical reactions without being consumed. Enzymes contain one or more active sites that...
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Catalytically Perfect Enzymes01:07

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The theory of catalytically perfect enzymes was first proposed by W.J. Albery and J. R. Knowles in 1976. These enzymes catalyze biochemical reactions at high-speed. Their catalytic efficiency values range from 108-109 M-1s-1. These enzymes are also called 'diffusion-controlled' as the only rate-limiting step in the catalysis is that of the substrate diffusion into the active site. Examples include triose phosphate isomerase, fumarase, and superoxide dismutase.
 
Most enzymes...
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Introduction to Mechanisms of Enzyme Catalysis01:13

Introduction to Mechanisms of Enzyme Catalysis

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For many years, scientists thought that enzyme-substrate binding took place in a simple "lock-and-key" fashion. This model stated that the enzyme and substrate fit together perfectly in one instantaneous step. However, current research supports a more refined view scientists call induced fit. The induced-fit model expands upon the lock-and-key model by describing a more dynamic interaction between enzyme and substrate. As the enzyme and substrate come together, their interaction causes...
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Conservative Site-specific Recombination and Phase Variation02:53

Conservative Site-specific Recombination and Phase Variation

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Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...
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Updated: Dec 8, 2025

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
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Split enzymes: Design principles and strategy.

Shion A Lim1, James A Wells1

  • 1Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, San Francisco, CA, United States.

Methods in Enzymology
|September 18, 2020
PubMed
Summary
This summary is machine-generated.

Researchers engineered split enzymes for precise control of biological processes. By fusing inactive enzyme fragments to inducible dimerization domains, enzyme activity is switched on with a small molecule, enabling new research capabilities.

Keywords:
Enzyme engineeringMolecular switchesProtein designProtein engineeringSplit enzymes

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Area of Science:

  • Biochemistry and Molecular Biology
  • Synthetic Biology
  • Enzyme Engineering

Background:

  • Precise control over enzymatic activity is crucial for understanding and manipulating biological processes.
  • Split enzymes, inactive fragments reconstituted by external stimuli, offer a method for switch-like control.
  • Inducible dimerization domains, responsive to small molecules, are key components in split enzyme design.

Purpose of the Study:

  • To summarize key design principles and strategies for engineering split enzymes.
  • To provide guidance for future efforts in developing novel split enzyme systems.
  • To illustrate these principles using examples of split enzymes developed by the research group.

Main Methods:

  • Designing split enzymes by separating active proteins into two inactive polypeptide fragments.
  • Fusing each fragment to inducible dimerization domains that mediate protein-ligand interactions.
  • Utilizing small molecules to trigger dimerization and reconstitute functional enzyme activity.

Main Results:

  • Demonstrated the successful engineering of split enzymes across various enzyme classes.
  • Showcased how split enzymes enable spatial and temporal control over biological processes.
  • Provided practical design principles and strategies applicable to future split enzyme development.

Conclusions:

  • Split enzymes represent powerful synthetic tools for dissecting complex biological functions.
  • The described design principles facilitate the creation of novel, controllable enzymatic systems.
  • This approach significantly advances the ability to manipulate and study biological systems with high precision.