Substrate independent ATPase activity may complicate high throughput screening.

Area of Science:

• Biochemistry
• Enzymology
• Drug Discovery

Background:

• High-throughput screening (HTS) often measures inorganic phosphate ([Pi]) release to identify enzyme inhibitors.
• SAICAR synthetase (PurC) is a key enzyme in the purine biosynthesis pathway, making it a target for drug discovery.

Purpose of the Study:

• To develop and validate a protocol for screening inhibitors of SAICAR synthetase (PurC) using [Pi] release.
• To investigate the source of observed [Pi] signals in control samples during PurC inhibition screening.

Main Methods:

• Enzymatic assay design for PurC inhibitor screening.
• Kinetic analysis of [Pi] release over time.
• Investigation of ATP hydrolysis as a potential source of [Pi].

Main Results:

• A gradual increase in [Pi] was observed in positive control samples during PurC screening.
• PurC-catalyzed hydrolysis of ATP (ATPase activity) was identified as a significant contributor to the observed [Pi] signal.
• This substrate-independent ATPase activity complicates the interpretation of [Pi] release in HTS assays.

Conclusions:

• Substrate-independent ATPase activity of PurC can lead to false positives or complicate the interpretation of [Pi] release in HTS assays.
• Assay protocols for PurC inhibitor screening must account for potential ATP hydrolysis to ensure accurate results.
• Understanding enzyme-specific ATPase activity is crucial for robust HTS assay development.