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A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates
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Mutagenesis of apyrase conserved region 1 alters the nucleotide substrate specificity

Riku Okuhata1, Yuki Otsuka, Takahide Tsuchiya

  • 1Department of Materials and Life Science; Faculty of Science and Technology; Sophia University; Tokyo, Japan.

Plant Signaling & Behavior
|March 9, 2013
PubMed

Insights

Mimosa pudica apyrases MP67 and MpAPY2 exhibit distinct substrate specificities. Amino acid substitutions in the conserved DXG motif altered their preference for ATP/ADP hydrolysis, highlighting the motif's role in nucleotide binding.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Mimosa pudica possesses two distinct apyrases, MP67 and MpAPY2, with differing substrate specificities.
  • The nucleotide binding motif (DXG) is conserved across apyrases and is crucial for enzyme function.

Purpose of the Study:

  • To investigate the role of the DXG motif in determining the substrate specificity of Mimosa pudica apyrases.
  • To elucidate how specific amino acid residues within the DXG motif influence ATP and ADP hydrolysis.

Main Methods:

  • Site-directed mutagenesis was employed to introduce single amino acid substitutions at position X within the DXG motif of MP67 and MpAPY2.
  • Enzyme kinetics, including the measurement of ATP/ADP hydrolysis rates and binding affinities, were used to characterize wild-type and mutant enzymes.

Main Results:

  • The S63A-MP67 mutant showed a significantly increased ratio of ATP/ADP hydrolysis velocity compared to wild-type MP67.
  • The A75S-MpAPY2 mutant exhibited enhanced binding affinity for ADP compared to its wild-type counterpart.
  • These findings indicate that the residue at position X in the DXG motif is critical for dictating nucleotide preference.

Conclusions:

  • The DXG motif's residue at position X is a key determinant of apyrase substrate specificity.
  • Understanding this mechanism provides insights into the molecular basis of nucleotide discrimination in enzymes.
  • This research contributes to the broader understanding of enzyme kinetics and protein structure-function relationships.

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