We monitored the hydrolysis of imidazole phosphate in the presence and absence of histidyls to determine whether or not the hydrolysis was catalysed (Figure , SI Section S2). First, we followed the uncatalysed background hydrolysis of imidazole phosphate using 31 P NMR spectroscopy with 50 mM calcium imidazole phosphate in 0.5 M MOPS buffer and 0.1 M citric acid and 50 mM HMPA internal standard at pH 7.5 and 22 °C (SI Section S2.10). The citric acid was added to prevent precipitation of calcium phosphate. Under these conditions, the imidazole phosphate (ImP) slowly hydrolysed to orthophosphate (Pi) over a period of 48 h (Figure , q and s traces). Formation of diphosphoimidazole from the reaction of imidazole phosphate with itself was also observed (SI Figure ), and this accounts for the difference in concentration between imidazole phosphate and orthophosphate in Figure (SI Figure ). To determine the reaction rate constants for the hydrolysis of imidazole phosphate, a reaction kinetics scheme was fitted to the experimental data (SI Figure and). The first order rate constant for the hydrolysis of imidazole phosphate was determined to be khyd,ImP = 3.9 × 10 -7 s -1 (SI Table ). The first order rate constant for the hydrolysis of diphosphoimidazole (DPI) was an order of magnitude larger at khyd,DPI = 8.6 × 10 -6 s -1 (SI Table ).