Metal stopping reagents facilitate discontinuous activity assays of the de novo purine biosynthesis enzyme PurE
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.
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This is the Author Accepted Manuscript of Kelly L. Sullivan, Loredana C. Huma, Elwood A. Mullins, Michael E. Johnson, T. Joseph Kappock (2014) Metal stopping reagents facilitate discontinuous activity assays of the de novo purine biosynthesis enzyme PurE, Analytical Biochemistry 452, 43–45. http://dx.doi.org/10.1016/j.ab.2014.02.004. It has been given a CC-BY-NC-ND licence