A Fluorescence-Based Assay for N5-Carboxyaminoimidazole Ribonucleotide Mutase |
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Authors: | Marcella F Sharma Dr Shiv K Sharma Dr Cale C Streeter Prof Steven M Firestine |
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Affiliation: | Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, 259 Mack Avenue, 48201 Detroit, MI, USA |
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Abstract: | The enzyme N5-carboxylaminoinidazole ribonucleotide (N5-CAIR) mutase is found in microbial de novo purine biosynthesis but is absent in humans making it an attractive antimicrobial target. N5-CAIR mutase catalyzes the synthesis of carboxyaminoimidazole ribonucleotide (CAIR) from N5-CAIR which is itself prepared from aminoimidazole ribonucleotide (AIR) by the enzyme N5-CAIR synthetase. During our research on identifying inhibitors of N5-CAIR mutase, we developed an innovative, fluorescence-based assay to measure the activity of this enzyme. This assay relies upon our recent serendipitous observation that AIR reversibly reacts with the compound isatin. Reaction of a fluorescently-tagged isatin with AIR resulted in a large increase in fluorescence intensity allowing a measurement of the concentration of AIR in solution. From this observation, we developed a reproducible, non-continuous assay that can replicate the known kinetic parameters of the enzyme and can readily detect a recognized inhibitor of the enzyme. This assay should find utility in screening for inhibitors targeting N5-CAIR mutase. |
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Keywords: | assay development enzyme inhibitors N5-CAIR mutase screening assays |
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