We next followed the hydrolysis of imidazole phosphate in the presence of 50 mM histidine under identical conditions to see if histidine catalysed the reaction. A representative set of 31 P NMR spectra over time are shown in Figure . At the start, the only phosphorylated species present in solution is imidazole phosphate (d 4.65 ppm). Over time, a phosphorylated histidine intermediate (d 4.92 ppm) is formed. We confirmed with 1 H 31 P HMBC NMR spectroscopy that this phosphate is located on the imidazolyl ring of the histidine residue (SI Figure ). Diphosphoimidazole (d 4.55 ppm) is also formed over time. The concentration of the hydrolysis product orthophosphate (d 2.10 ppm) increases over time. In the presence of the histidine the hydrolysis reaction was accelerated compared to the uncatalysed reaction as seen in both the accelerated disappearance of imidazole phosphate and the accelerated appearance of orthophosphate (Figure , n and o traces). A reaction kinetics scheme for the histidyl catalysed hydrolysis of imidazole phosphate was again fitted to the experimental data (SI Figure and 20). The first order rate constant for the hydrolysis of the phosphorylated histidine intermediate (PHis) was determined to be khyd,PHis = 1.4 × 10 -5 s -1 (SI Table ) -a value that is approximately two orders of magnitude higher than that of the background hydrolysis of imidazole phosphate. This therefore confirmed that histidine catalysed the hydrolysis reaction.