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Related Concept Videos

Catalytically Perfect Enzymes01:07

Catalytically Perfect Enzymes

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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.
 
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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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Introduction to Mechanisms of Enzyme Catalysis01:13

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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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Enzymes speed up reactions by lowering the activation energy of the reactants. The speed at which the enzyme turns reactants into products is called the rate of reaction. Several factors impact the rate of reaction, including the number of available reactants. Enzyme kinetics is the study of how an enzyme changes the rate of a reaction.
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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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The organelle-specific signaling sequences direct proteins synthesized in the cytosol to their final destination like ER, mitochondria, peroxisomes, etc. Some of the proteins directed to ER are then trafficked via vesicles to other organelles within the cell or the extracellular environment through the Golgi complex. For example, the rough ER synthesizes soluble proteins for transportation to the lysosomes or secretion out of the cell. It can also synthesize transmembrane proteins that can...
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A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes
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Directed evolution improves the catalytic efficiency of TEV protease.

Mateo I Sanchez1,2, Alice Y Ting3,4

  • 1Departments of Genetics, Biology and Chemistry, Stanford University, Stanford, CA, USA.

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|December 11, 2019
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Researchers engineered faster Tobacco etch virus protease (TEV) using yeast-directed evolution. This breakthrough enhances protease activity for biotechnology applications, improving neuronal activity recording.

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Area of Science:

  • Biotechnology and Molecular Biology
  • Enzyme Engineering and Directed Evolution

Background:

  • Tobacco etch virus protease (TEV) is crucial in biotechnology due to its high sequence specificity.
  • A significant limitation of TEV is its inherently slow catalytic rate, hindering broader applications.
  • Existing methods lack efficient ways to improve protease catalytic efficiency.

Purpose of the Study:

  • To develop a novel yeast-based platform for the directed evolution of protease catalytic properties.
  • To engineer enhanced variants of TEV protease with improved catalytic rates.
  • To demonstrate the utility of evolved TEV in sensitive biological measurements.

Main Methods:

  • A yeast-based directed evolution system was established to screen for improved protease activity.
  • Protease activity was measured by the release of a membrane-anchored transcription factor.
  • A photosensory LOV domain was used to control substrate accessibility, enabling selection for faster variants under decreasing light exposure.

Main Results:

  • A yeast-based directed evolution platform was successfully developed and implemented.
  • The TEV-S153N mutant (uTEV1Δ) demonstrated significantly enhanced catalytic properties.
  • Incorporation into the FLARE system improved signal/background by 27-fold, enabling neuronal activity recording with 60-s resolution.

Conclusions:

  • The developed directed evolution platform is a generalizable method for improving protease function.
  • Engineered TEV variants, like uTEV1Δ, offer substantial improvements over the wild-type enzyme.
  • These advancements hold significant potential for diverse biotechnology and neuroscience applications.