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Reduced activity of bamhi variants c54i, c64w, and c54d/c64r is consistent with the substrate-assisted catalysis

A S Acharya1, K B Roy

  • 1Centre for Biotechnology, Jawaharlal Nehru University, New Delhi, 110067, India.

Biochemical and Biophysical Research Communications
|September 11, 2001
PubMed

Insights

Investigating restriction endonuclease BamHI mutants revealed that cysteine residues at positions 54 and 64 significantly impact enzyme activity and substrate binding. Mutations altered enzyme kinetics and protein structure, suggesting roles in catalysis and substrate interaction.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Restriction endonuclease BamHI is a key enzyme in molecular biology.
  • The roles of specific cysteine residues in BamHI catalysis remain incompletely understood.

Purpose of the Study:

  • To investigate the function of cysteine residues at positions 54 and 64 in BamHI enzyme activity.
  • To characterize the kinetic and structural effects of specific BamHI mutations.

Main Methods:

  • Site-directed mutagenesis (megaprimer PCR) to generate C54I, C54W, and C54D:C64R mutants.
  • Gene cloning, sequencing, protein expression, and purification.
  • Kinetic parameter determination (Km, Kcat) using synthetic oligonucleotide substrates.
  • Circular dichroism (CD) spectroscopy and melting curve analysis for structural assessment.

Main Results:

  • All generated BamHI mutants exhibited higher Km values than wild-type, indicating reduced substrate affinity.
  • The C54W mutant showed altered CD spectra, suggesting changes in protein secondary structure.
  • The double mutant C54D:C64R displayed reduced catalytic activity, consistent with substrate-assisted catalysis.

Conclusions:

  • Cysteine 54 mutations in BamHI likely affect enzyme activity by perturbing local protein structure.
  • The reduced activity of the double mutant supports a substrate-assisted catalysis mechanism for BamHI.

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