Protein histidine phosphorylation plays a vital role in cell signaling and metabolic processes, and phosphohistidine (pHis) phosphatases such as protein histidine phosphatase 1 (PHPT1) and LHPP have been linked to cancer and diabetes, making them novel drug targets and biomarkers. Unlike the case for other classes of phosphatases, further studies of PHPT1 and other pHis phosphatases have been hampered by the lack of specific activity assays in complex biological mixtures. Previous methods relying on radiolabeling are hazardous and technically laborious, and small-molecule phosphatase probes are not selective toward pHis phosphatases. To address these issues, we herein report a fluorescent probe based on chelation-enhanced fluorescence (CHEF) to continuously measure the pHis phosphatase activity of PHPT1. Our probe exhibited excellent sensitivity and specificity toward PHPT1, enabling the first specific measurement of PHPT1 activity in cell lysates. Using this probe, we also obtained more physiologically relevant kinetic parameters of PHPT1, overcoming the limitations of previously used methods.
Keywords: PHPT1; chelation-enhanced fluorescence; enzyme kinetics; fluorescent probe; phosphohistidine phosphatase.