title: Metal stopping reagents facilitate discontinuous activity assays of the de novo purine biosynthesis enzyme PurE.
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 24h, 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.
authors: Department of Biochemistry, Purdue University, West Lafayette, IN 47907-2063, USA.
authors: Kelly L
authors: KL
authors: Sullivan
authors: Center for Pharmaceutical Biotechnology and Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, IL 60607-7173, USA.
authors: Loredana C
authors: LC
authors: Huma
authors: Department of Biochemistry, Purdue University, West Lafayette, IN 47907-2063, USA; Department of Chemistry, Washington University in St. Louis, St. Louis, MO 63130-4899, USA.
authors: Elwood A
authors: EA
authors: Mullins
authors: Center for Pharmaceutical Biotechnology and Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, IL 60607-7173, USA.
authors: Michael E
authors: ME
authors: Johnson
authors: Department of Biochemistry, Purdue University, West Lafayette, IN 47907-2063, USA. Electronic address: kappock@purdue.edu.
authors: T Joseph
authors: TJ
authors: Kappock
mesh_terms: D057075:Enzyme Assays / Q000379:methods*; D057166:High-Throughput Screening Assays; D007535:Isomerases / Q000737:chemistry* / Q000378:metabolism*; D011687:Purines / Q000096:biosynthesis*; D012265:Ribonucleotides / Q000378:metabolism; D019287:Zinc Sulfate / Q000737:chemistry*
chemical_list: D011687:Purines; D012265:Ribonucleotides; D019287:Zinc Sulfate; D007535:Isomerases; C030985:purine
keywords: Aminoimidazole; Chelation; Purine biosynthesis; Substrate depletion