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Assaying Protein Kinase Activity with Radiolabeled ATP
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Binding interactions in a kinase active site modulate background ATP hydrolysis.

Yun Wang1, Grace Hanrahan1, Frederic Abou Azar1

  • 1Department of Chemistry, McGill University, 801 Sherbrooke St. W., Montreal, Quebec H3A 0B8, Canada.

Biochimica Et Biophysica Acta. Proteins and Proteomics
|October 1, 2021
PubMed
Summary

Non-substrate molecules significantly enhance ATP hydrolysis by aminoglycoside-3'-phosphotransferase-IIIa (APH(3')-IIIa), revealing substrate-induced catalytic activation mechanisms crucial for enzyme function.

Keywords:
APH(3′)-IIIaAminoglycoside-3′-phosphotransferase-IIIaIsothermal titration CalorimetryPre-steady state kineticsSingle turnover kineticsTheorell-chance mechanism

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Area of Science:

  • Biochemistry
  • Enzymology
  • Molecular Biology

Background:

  • Kinases catalyze phosphate transfer from nucleoside triphosphates (NTPs) to substrates.
  • Kinases can also hydrolyze NTPs, a less efficient reaction consuming cellular energy.
  • Substrate binding is hypothesized to activate NTP hydrolysis, but functional data are scarce.

Purpose of the Study:

  • To investigate substrate-induced activation of ATP hydrolysis by aminoglycoside-3 -phosphotransferase-IIIa (APH(3 )-IIIa).
  • To characterize the kinetics and biophysical mechanisms of APH(3 )-IIIa's catalytic activity.
  • To explore the potential for designing small-molecule effectors targeting bacterial resistance enzymes.

Main Methods:

  • Utilized isothermal titration calorimetry (ITC) kinetics methods.
  • Measured kinetic parameters (K , k , product inhibition constants) and single-turnover kinetics.
  • Assessed the impact of non-substrate aminoglycosides (nsAmgs) on ATP hydrolysis.

Main Results:

  • Non-substrate aminoglycosides (nsAmgs) increased the rate of ATP γ-phosphate cleavage by 10- to 20-fold under single-turnover conditions.
  • Demonstrated that substrate-like interactions enhance catalytic rates, supporting substrate-induced activation.
  • Obtained detailed kinetic data on nsAmg and ADP binding, elucidating the enzyme's Theorell-Chance mechanism.

Conclusions:

  • Enzyme-effector interactions significantly enhance catalytic activity, linking substrate binding to increased hydrolysis rates.
  • Kinetic and biophysical data provide insights into the mechanism of APH(3 )-IIIa.
  • Findings suggest strategies for designing small molecules to inhibit antibiotic resistance enzymes by triggering efficient ATP hydrolysis.