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
Purine biosynthesis; Aminoimidazole; Substrate depletion; Chelation
Date of this Version
Sullivan, Kelly L.; Huma, Loredana C.; Mullins, Elwood; Johnson, Michael E.; and Kappock, T. Joseph, "Metal stopping reagents facilitate discontinuous activity assays of the de novo purine biosynthesis enzyme PurE" (2014). Department of Biochemistry Faculty Publications. Paper 12.