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Measuring In Vitro ATPase Activity for Enzymatic Characterization
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Kinetics of ATP/ADP binding to the gp16 ATPase

Aaron Morgan1, Allen Eastlund2, Christopher Fischer1

  • 1Department of Physics and Astronomy, University of Kansas, Lawrence, Kansas.

Biophysical Journal
|April 14, 2022
PubMed

Insights

The gp16 ATPase binds ATP with high affinity and ADP with low affinity, revealing insights into the Bacillus subtilis Φ29 bacteriophage DNA translocation motor mechanism.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Structural Biology

Background:

  • The Bacillus subtilis Φ29 bacteriophage utilizes a pentameric gp16 ATPase motor for double-stranded DNA (dsDNA) translocation.
  • The precise mechanism by which gp16 subunits couple ATP hydrolysis to dsDNA movement is not fully understood.

Purpose of the Study:

  • To investigate the nucleotide binding properties of monomeric gp16.
  • To elucidate the binding affinities of ATP and ADP to gp16.

Main Methods:

  • Stopped-flow fluorescence spectroscopy was employed to monitor the binding of fluorophore-labeled ATP and ADP to monomeric gp16.
  • Analysis of binding kinetics and affinities.

Main Results:

  • Monomeric gp16 binds ATP via a single-step mechanism with an affinity of 523.8 ± 247.3 nM.
  • The binding affinity for ADP is significantly lower, with a lower limit of 30 μM.
  • The observed differences in binding affinities are energetically consistent with a cyclical binding, hydrolysis, and release process.

Conclusions:

  • The distinct binding affinities for ATP and ADP provide crucial data for understanding the energy transduction mechanism of the gp16 motor.
  • These findings are a foundational step towards modeling inter-subunit communication within the pentameric gp16 motor complex.

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