Abstract
Inorganic phosphate release, [Pi], is often measured in an enzymatic reaction in a high throughput setting. Based on the published mechanism, we designed a protocol for our screening for inhibitors of SAICAR synthetase (PurC), and we found a gradual increase in [Pi] in positive control samples over the course of the day. Further investigation indicated that hydrolysis of ATP catalyzed by PurC, rather than substrate-related phosphate release, was responsible for a partial contribution to the signals in the control samples. Thus substrate-independent ATPase activity may complicate high throughput screening.
Highlights
Inorganic phosphate release, [Pi], is often measured in an enzymatic reaction in a high throughput setting
We implemented a Malachite Green assay (MGA) in a high throughput screening (HTS) setup to identify compounds that inhibit the activity of Bacillus anthracis PurC (BaPurC)
MGA has long been used to monitor the activities of enzymes that release inorganic phosphate by detecting the phosphomolybdate-Malachite Green complex [7,8], and is a common method for the primary screening of inhibitors against enzymes with NTPase activity [9,10,11]
Summary
Inorganic phosphate release, [Pi], is often measured in an enzymatic reaction in a high throughput setting. Keywords Malachite Green; high throughput screening; substrate-independent ATPase activity As an individual enzyme in many bacterial species, PurC converts L-Asp, 4-carboxy-5-aminoimidazole ribonucleotide (CAIR) and ATP to 4-(N-succino)-5aminoimidazole-4-carboxamide ribonucleotide (SAICAR), ADP and Pi [1,2,3].
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