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The Due Ferri single chain (DFsc) family of proteins was originally created to better understand the characteristics of carboxylate-bridged diiron proteins. It was observed that when zinc coordinates to the active site of this protein rather than iron, the protein cleaves phosphate bonds via hydrolysis. To better understand, and potentially enhance this reactivity, mutations have been made in or near the metal binding active site. In this project, we have developed an assay using phosphorus nuclear magnetic resonance spectroscopy (31P-NMR) to monitor the catalytic activity of DFsc by observing the conversion of bis(4-nitrophenyl) phosphate (BNPP) to para-nitrophenolate (PNP) and para-nitro-phenylphosphate (pNPP). Using integral calculations, rates of reaction for DFsc and its mutants were determined. Additionally, the developed 31P-NMR protocol was replicated on a UV-Vis plate reader. The NMR protocol indicated that DFsc is the most efficient protein for phosphatase activity, however, the UV-Vis data indicated that 3-HisG4DFsc is the most efficient protein for phosphatase activity. Further experimentation is necessary to further optimize these protocols and acquire more consistent data that can be compared across different approaches.
Bender, Alex, "Characterization of DFsc Phosphatase Activity" (2022). Chemistry Summer Fellows. 41.
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