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Related Experiment Videos

EcoRI variant N199H has enhanced specific activity

J J Kim1, K T Min, M H Kim

  • 1Korea Research Institute of Biotechnology and Bioscience, K.I.S.T., Yusong, Taejon, South Korea.

Gene
|May 24, 1996
PubMed
Summary

Researchers modified the Asn199 residue in EcoRI restriction endonuclease. The N199H variant exhibited enhanced enzyme activity, suggesting this residue

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Area of Science:

  • Molecular Biology
  • Enzymology
  • Protein Engineering

Background:

  • EcoRI restriction endonuclease is a key enzyme in molecular biology.
  • The Asn199 residue's role in EcoRI's function is not fully understood.
  • Investigating specific residues can elucidate enzyme mechanisms.

Purpose of the Study:

  • To determine the role of the Asn199 residue in EcoRI.
  • To assess if Asn199 is involved in nucleotide recognition or catalysis.
  • To engineer EcoRI variants with altered or improved activity.

Main Methods:

  • Site-directed mutagenesis was used to create EcoRI variants with amino acid substitutions at position 199.
  • Enzyme activity was measured by assessing the relative cleavage rates of the generated variants.

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  • Specific activity was compared between wild-type EcoRI and its variants.

    • Main Results:

    • Several EcoRI variants were successfully generated, including N199D, N199H, N199L, N199R, N199S, and N199V.
    • The relative cleavage rates varied significantly among the variants.
    • The N199H variant demonstrated approximately twofold higher specific activity compared to wild-type EcoRI.
    • The order of cleavage activity was N199H > wt > N199L > N199V > N199S > N199R > N199D.

    Conclusions:

    • The Asn199 residue plays a role in modulating EcoRI's catalytic activity.
    • Substitution with Histidine (N199H) enhances EcoRI's specific activity.
    • Further studies are warranted to fully elucidate the mechanism of nucleotide recognition and catalysis involving Asn199.