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

Authors

Kelly L. Sullivan, Purdue UniversityFollow
Loredana C. Huma, University of Illinois at ChicagoFollow
Elwood Mullins, Purdue University
Michael E. Johnson, University of Illinois at ChicagoFollow
T. Joseph Kappock, Purdue UniversityFollow

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

Comments

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

Keywords

Purine biosynthesis; Aminoimidazole; Substrate depletion; Chelation

Date of this Version

5-1-2014

DOI

10.1016/j.ab.2014.02.004

Recommended Citation

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.
http://dx.doi.org/10.1016/j.ab.2014.02.004