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.
In contrast to well‐recognized protein phosphorylation on the side‐chain oxygen of Ser, Thr, or Tyr residues, analogous phosphoramidation of the nitrogen of His, Lys, and Arg side chains remains much less investigated, mainly due to the instability of post‐translational modifications and technical difficulties involved in their analysis. For example, reports on the enzyme activities responsible for the formation and hydrolysis of these phosphoramidates date back to as early as the 1950s, but some of these enzymes have only recently been identified and functionally characterized; this has been aided by the development of novel research tools. In this review, we summarize current knowledge of the enzymes that hydrolyze protein N‐phosphoramidates, in terms of their structure, activities, and biological functions, as well as the chemical tools used to investigate them.
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