Kinetics of ATP/ADP binding to the gp16 ATPase

Aaron Morgan, Allen Eastlund, Christopher Fischer, Paul Jardine

Research output: Contribution to journalArticlepeer-review


The gp16 ATPase is the constituent subunit of the pentameric dsDNA (double-stranded deoxyribonucleic acid) translocation motor of the Bacillus subtilis Φ29 bacteriophage. Although recent single-molecule studies have provided tantalizing clues about the activity of this motor, the mechanism by which the gp16 subunits couple the energy obtained from the binding and hydrolysis of ATP to the mechanical work of dsDNA translocation remains unknown. To address this need, we have characterized the binding of fluorophore-labeled ATP and ADP to monomeric gp16 using a stopped-flow fluorescence assay. These experiments show that the binding of ATP/ADP occurs through a single-step mechanism with corresponding affinities of 523.8 ± 247.3 nM for ATP and a lower limit of 30 μM for ADP. When analyzed through the lens of changes in free energy of the system, this difference in binding affinities is reasonable for a cyclical process of binding, hydrolysis, and product release. In addition to answering questions about the activity of monomeric gp16, these results are also a necessary step in constructing a model for intersubunit communication within the pentameric gp16 motor.

Original languageEnglish (US)
Pages (from-to)1909-1918
Number of pages10
JournalBiophysical journal
Issue number10
StatePublished - May 17 2022

Bibliographical note

Publisher Copyright:
© 2022 Biophysical Society


  • Adenosine Diphosphate/metabolism
  • Adenosine Triphosphatases/metabolism
  • Adenosine Triphosphate/metabolism
  • Bacillus Phages/genetics
  • DNA, Viral/metabolism
  • Hydrolysis
  • Kinetics

PubMed: MeSH publication types

  • Journal Article


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