Metal stopping reagents facilitate discontinuous activity assays of the de novo purine biosynthesis enzyme PurE |
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| Authors: | Kelly L. Sullivan Loredana C. Huma Elwood A. Mullins Michael E. Johnson T. Joseph Kappock |
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| Affiliation: | 1. Department of Biochemistry, Purdue University, West Lafayette, IN 47907-2063, USA;2. Center for Pharmaceutical Biotechnology and Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, IL 60607-7173, USA;3. Department of Chemistry, Washington University in St. Louis, St. Louis, MO 63130-4899, USA |
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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. |
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| Keywords: | Purine biosynthesis Aminoimidazole Substrate depletion Chelation |
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