Figure 2.

Substrate specificity of CthPnkp. Reaction mixtures (25 µl) containing 50 mM Tris–HCl (pH 7.5), 0.5 mM MnCl₂, 1.5 µg of CthPnkp and 10 mM substrate as specified were incubated for 30 min at 45°C. The reactions were quenched by adding 20 mM EDTA and then 0.9 ml of 1 M Na₂CO₃. Release of p-nitrophenol was determined by measuring A₄₁₀ and interpolating the value to a p-nitrophenol standard curve. The extents of formation of p-nitrophenol are plotted at left. The chemical structures of the substrates bis-p-nitrophenyl phosphate ①, p-nitrophenyl phenylphosphonate ②, dimethyl-p-nitrophenyl phosphate ③, thymidine 5′-monophosphate-p-nitrophenyl ester ④ and p-nitrophenyl phosphorylcholine ⑤ are shown at right.