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Elsevier, Analytical Biochemistry, (452), p. 43-45, 2014

DOI: 10.1016/j.ab.2014.02.004

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Metal stopping reagents facilitate discontinuous activity assays of the de novo purine biosynthesis enzyme PurE

This paper is available in a repository.
This paper is available in a repository.

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Abstract

The conversion of 5-aminoimidazole ribonucleotide (AIR) to 4-carboxy-AIR (CAIR) represents an unusual divergence in purine biosynthesis: microbes and nonmetazoan eukaryotes use class I PurEs while animals use class II PurEs. Class I PurEs are therefore a potential antimicrobial target; however, no enzyme activity assay is suitable for high throughput screening (HTS). Here we report a simple chemical quench that fixes the PurE substrate/product ratio for 24 h, as assessed by the Bratton-Marshall assay (BMA) for diazotizable amines. The ZnSO4 stopping reagent is proposed to chelate CAIR, enabling delayed analysis of this acid-labile product by BMA or other HTS methods.