Fig. 1.

OA-NO₂ inhibits PknG kinase activity. (A) GarA phosphorylation by PknG. Linear MALDI-TOF mass spectrum of GarA (m/z 17145, dashed line), GarA phosphorylated by PknG using a molar ratio PknG:GarA of 1:20 (m/z 17222, gray line), and GarA phosphorylated by PknG pretreated with 50 μM OA-NO₂ for 10 min (m/z 17143, black line). (B) PknG autophosphorylation. Upper panel: linear MALDI-TOF spectrum of native PknG after tryptic digestion showing basal autophosphorylation pattern. Unphosphorylated peptide with seq. 10–60 (m/z 5395.8), monophosphorylated (m/z 5475.4), and diphosphorylated species (m/z 5555.1) are detected as expected. Middle panel: linear MALDI-TOF spectrum of PknG after incubation with ATP and Mn²⁺ and further typtic digestion (autophoshorylation positive control). Diphosphorylated peptide (m/z 5555.0) is the most intense ion observed while unphosphorylated and monophosphorylated ions were almost undetectable. Lower panel: linear MALDI-TOF spectrum of tryptic digestion of PknG treated with 30 μM OA-NO₂ for 10 min before autophosporylation reaction. The detection of all three forms of the phosphorylatable peptide indicates that OA-NO₂ impairs conversion into the fully phosphorylated species. The spectra are representative of three independent experiments. (C) Dose-dependent inhibition of PknG by OA-NO₂. PknG (8 μM) was incubated with OA-NO₂ ranging from 0 to 80 μM in 70 mM ammonium bicarbonate, pH 8.0, at 25 °C. As a control, PknG was incubated with vehicle under the same experimental conditions. After 10 min of incubation enzyme activities were determined. Samples were analyzed in triplicates. (D) Time-dependent inhibition of PknG by OA-NO₂. PknG (10 μM) was incubated with 50 μM OA-NO₂ (treated PknG) or vehicle (control PknG) in 70 mM ammonium bicarbonate, pH 8.0, at 25 °C. At the indicated time points aliquots of control and treated PknG were removed, and enzyme inhibition was determined using Kinase Glo® by comparison with control activity. Samples were analyzed in triplicates.