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[Barnase mutant Ser57Ala: preparation and properties]

E Iu Kolbanovskaia1, A L Okorokov, K I Panov

  • 1National Institute of Health, Bethesda, Md. 20892.

Molekuliarnaia Biologiia
|May 1, 1994
PubMed
Summary

Researchers investigated barnase, an enzyme that breaks down RNA. Mutating a specific amino acid (Ser-57) in barnase did not significantly alter its RNA hydrolysis kinetics, suggesting this residue is not critical for polynucleotide cleavage.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Barnase is an extracellular ribonuclease from Bacillus amyloliquefaciens.
  • It belongs to a family of small microbial ribonucleases with conserved structures and functions.
  • Barnase hydrolyzes phosphodiester bonds in RNA, primarily at guanosine nucleotides.

Purpose of the Study:

  • To investigate the molecular basis of barnase's guanylic specificity.
  • To determine the role of Ser-57 in the "base recognition loop" of barnase.
  • To analyze the effect of Ser-57 mutation on the enzyme's kinetic properties.

Main Methods:

  • Site-directed mutagenesis was used to replace Ser-57 with Alanine in barnase.
  • The mutant barnase protein was expressed in an Escherichia coli system.

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  • Kinetic parameters of polynucleotide hydrolysis by the mutant enzyme were studied.

    • Main Results:

    • Mutation of Ser-57 to Alanine in the "base recognition loop" of barnase was successfully created.
    • The mutant barnase protein was purified and characterized.
    • The kinetic parameters for the hydrolysis of polynucleotide substrates by the Ser-57 mutant were not significantly affected.

    Conclusions:

    • The Ser-57 residue in the "base recognition loop" of barnase is not essential for the catalytic hydrolysis of polynucleotide substrates.
    • The guanylic specificity of barnase in polynucleotide hydrolysis may not be solely dependent on this specific residue.
    • Further studies are needed to elucidate the precise molecular mechanisms underlying barnase's substrate specificity.