Abstract
Although the importance of protein histidine phosphorylation in mammals has been a subject of increasing interest, few chemical probes are available for monitoring and manipulating PHP activity. Here, we present an optimized and validated protocol for assaying the activity of PHPT1 using the fluorogenic substrate DiFMUP. The kinetic parameters of our optimized assay are significantly improved as compared with other PHPT1 assays in the literature, with a kcat of 0.39 ± 0.02 s-1, a Km of 220 ± 30 μM, and a kcat/ Km of 1800 ± 200 M-1 s-1. In addition, the assay is significantly more sensitive as a result of using a fluorescent probe, requiring only 109 nM enzyme as compared with 2.4 μM as required by previously published assays. In the process of assay optimization, we discovered that PHPT1 is sensitive to a reducing environment and inhibited by transition-metal ions, with one apparent Cu(II) binding site with IC50 value of 500 ± 20 μM and two apparent Zn(II) binding sites with IC50 values of 25 ± 1 and 490 ± 20 μM.
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