Related Experiment Video
Updated: May 2, 2026

Measuring In Vitro ATPase Activity for Enzymatic Characterization
Published on: August 23, 2016
Lytic water dynamics reveal evolutionarily conserved mechanisms of ATP hydrolysis by TIP49 AAA+ ATPases
Arina Afanasyeva1, Angela Hirtreiter2, Anne Schreiber2
1Department of Biophysics, Saint Petersburg State Polytechnical University, Saint Petersburg 195251, Russia; Division of Molecular and Radiation Biophysics, Petersburg Nuclear Physics Institute, Gatchina 188300, Russia.
Abstract:
Eukaryotic TIP49a (Pontin) and TIP49b (Reptin) AAA+ ATPases play essential roles in key cellular processes. How their weak ATPase activity contributes to their important functions remains largely unknown and difficult to analyze because of the divergent properties of TIP49a and TIP49b proteins and of their homo- and hetero-oligomeric assemblies. To circumvent these complexities, we have analyzed the single ancient TIP49 ortholog found in the archaeon Methanopyrus kandleri (mkTIP49). All-atom homology modeling and molecular dynamics simulations validated by biochemical assays reveal highly conserved organizational principles and identify key residues for ATP hydrolysis. An unanticipated crosstalk between Walker B and Sensor I motifs impacts the dynamics of water molecules and highlights a critical role of trans-acting aspartates in the lytic water activation step that is essential for the associative mechanism of ATP hydrolysis.
Related Concept Videos
ATP Driven Pumps I: An Overview
There are four main types of ATP-driven pumps - P-type, V-type, F-type, and ABC transporter. All these pumps are of varying complexities and...
ATP Driven Pumps III: V-type Pumps
The peripheral or cytosolic V1 domain with eight subunits is involved in ATP hydrolysis. The integral or transmembrane V0 domain containing at least five subunits...
Hydrolysis of ATP
If one phosphate group is removed, a molecule of ADP—adenosine diphosphate—remains, along with inorganic phosphate. ADP can be further hydrolyzed to AMP—adenosine...
Hydrolysis of ATP
ATP Synthase: Mechanism
ATP Synthase: Structure

