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Related Concept Videos

ATP Energy Storage and Release01:31

ATP Energy Storage and Release

ATP is a highly unstable molecule. Unless quickly used to perform work, ATP spontaneously dissociates into ADP and inorganic phosphate (Pi), and the free energy released during this process is lost as heat. The energy released by ATP hydrolysis is used to perform work inside the cell and depends on a strategy called energy coupling. Cells couple the exergonic reaction of ATP hydrolysis with endergonic reactions, allowing them to proceed.
One example of energy coupling using ATP involves a...
Phosphorylation01:02

Phosphorylation

The addition or removal of phosphate groups from proteins is the most common chemical modification that regulates cellular processes. These modifications can affect the structure, activity, stability, and localization of proteins within cells as well as their interactions with other proteins.
During phosphorylation, protein kinases transfer the terminal phosphate group of ATP to specific amino acid side chains of substrate proteins. Serine, threonine, and tyrosine are the most commonly...

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Related Experiment Video

Updated: Jun 17, 2026

Measuring In Vitro ATPase Activity for Enzymatic Characterization
07:38

Measuring In Vitro ATPase Activity for Enzymatic Characterization

Published on: August 23, 2016

High throughput calorimetry for evaluating enzymatic reactions generating phosphate.

Lieve Hoflack1, Manu De Groeve, Tom Desmet

  • 1Laboratory of Industrial Microbiology and Biocatalysis, Department of Biochemical and Microbial Technology, Faculty of Bioscience Engineering, Ghent University, Coupure links 653, B-9000 Ghent, Belgium. Lieve.Hoflack@UGent.be

Combinatorial Chemistry & High Throughput Screening
|December 18, 2009
PubMed
Summary

A new calorimetric assay enables high-throughput screening of enzymes producing inorganic phosphate. This method efficiently analyzes enzyme libraries for improved activity and promiscuity.

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Published on: December 19, 2011

Area of Science:

  • Biochemistry
  • Enzyme kinetics
  • High-throughput screening

Background:

  • Enzyme screening is crucial for discovering novel biocatalysts.
  • Existing phosphate assays can be limiting for large-scale screening.
  • Cellobiose phosphorylase (EC 2.4.1.20) is a key enzyme for disaccharide synthesis.

Purpose of the Study:

  • To develop and validate a novel calorimetric assay for high-throughput screening of enzymes that generate inorganic phosphate.
  • To assess the utility of this assay for enzyme library screening and substrate promiscuity studies.

Main Methods:

  • A calorimetric method was established to measure inorganic phosphate production.
  • The assay couples enzyme-generated phosphate to pyruvate oxidase and catalase reactions.
  • Simultaneous analysis of 48 reactions was performed in microtiter plate format.

Main Results:

  • The assay demonstrated a coefficient of variation of 3.14%, indicating high reproducibility.
  • Validation was achieved through comparison with a standard colorimetric phosphate assay.
  • The method successfully tested cellobiose phosphorylase for synthesizing rare disaccharides.

Conclusions:

  • The developed calorimetric assay is a robust and efficient tool for high-throughput enzyme screening.
  • This assay facilitates the identification of enzymes with enhanced activity and promiscuous substrate capabilities.
  • The method is suitable for screening enzyme and substrate libraries in biochemical research.