Figure 4.

Hydrolysis studies of phosphorylated HK853 species at various pH values. HK853 (2 μM) was incubated with Probe O or Probe S for 15 min. After 3 h of degradation, the media were neutralized, and TAMRA-N₃ was conjugated by CuAAC to the propargyl handle (30 min). Formation of pHis was quantified by fluorescence gel-based analysis. HK853 (5 μM) was incubated for 30 s with [γ−33P]-ATP (60 μM). After 3 h of degradation, the media was neutralized. Formation of pHis was quantified by gel-based analysis and phosphorimaging. Samples that did not undergo acidification or the 3 h incubation period were “100% phosphorylated” controls, to which all samples were compared. Plot indicates the percent of phosphorylated HK remaining in comparison to the 100% control for each probe or ATP as appropriate. n ≥ 3, error bars show standard deviation. Statistical analysis to compare two substrates at a specific pH performed with an unpaired t test (**p ≤ 0.01, *p ≤ 0.05).