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Related Concept Videos

Cofactors and Coenzymes01:24

Cofactors and Coenzymes

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Enzymes are proteins made of amino acids. The functional group of each constituent amino acid catalyzes a wide variety of chemical reactions via ionic interactions or acid-base reactions. However, amino acids cannot catalyze oxidation-reduction and group transfer reactions and need to be aided by non-protein components called cofactors. Cofactors are also referred to as the chemical teeth of an enzyme.
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Cofactors and Coenzymes01:27

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Cofactors and Coenzymes01:27

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Enzymes require additional components for proper function. There are two such classes of molecules: cofactors and coenzymes. Cofactors are metallic ions and coenzymes are non-protein organic molecules. Both of these types of helper molecule can be tightly bound to the enzyme or bound only when the substrate binds.
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Enzyme Kinetics01:19

Enzyme Kinetics

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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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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.
 
Most enzymes...
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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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Expression and Purification of Nuclease-Free Oxygen Scavenger Protocatechuate 3,4-Dioxygenase
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An Evolved Orthogonal Enzyme/Cofactor Pair.

Evan W Reynolds1, Matthew W McHenry1, Fabien Cannac1

  • 1Department of Chemistry, University of North Carolina-Chapel Hill , 125 South Road, CB 3290, Chapel Hill, North Carolina 27599, United States.

Journal of the American Chemical Society
|August 31, 2016
PubMed
Summary
This summary is machine-generated.

Researchers developed an orthogonal enzyme/heme pair strategy to expand hemoprotein functionality. A modified cytochrome P450 selectively uses a non-natural cofactor, enabling new chemical reactions.

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Area of Science:

  • Biochemistry
  • Biotechnology
  • Enzyme Engineering

Background:

  • Hemoproteins are crucial enzymes with heme cofactors.
  • Expanding hemoprotein functionality requires novel cofactor incorporation strategies.
  • Cytochrome P450s are versatile enzymes often engineered for new functions.

Purpose of the Study:

  • To develop an orthogonal enzyme/heme pair strategy for expanding hemoprotein functionality.
  • To engineer a cytochrome P450 enzyme (P450BM3) for selective incorporation of a non-native heme derivative.
  • To demonstrate the utility of the engineered enzyme/cofactor pair in catalysis.

Main Methods:

  • Exploited the promiscuous heme transport protein ChuA for importing heme derivatives.
  • Evolved P450BM3 for selective binding of iron deuteroporphyrin IX (Fe-DPIX) over endogenous heme.
  • Utilized X-ray crystallography to elucidate structural basis of cofactor selectivity.
  • Tested the catalytic activity of the orthogonal pair in carbenoid-mediated cyclopropanation.

Main Results:

  • Engineered P450BM3 selectively incorporated Fe-DPIX in the presence of native heme.
  • Structural analysis revealed mutations conferring selectivity through steric and binding interactions.
  • The evolved enzyme/cofactor pair demonstrated activity in non-natural olefin cyclopropanation.
  • Successfully generated an orthogonal enzyme/cofactor pair.

Conclusions:

  • The developed strategy enables the creation of orthogonal enzyme/cofactor pairs.
  • This approach significantly expands cofactor diversity for artificial metalloenzymes.
  • The engineered system offers a platform for novel biocatalytic applications.
  • Methodology promises broader applications in enzyme engineering and synthetic biology.