Here the disappearance of imidazole phosphate was accelerated and a phosphorylated acetyl histidine imidazolyl was formed (SI Figure ). However, no acceleration in the formation of orthophosphate was observed and the phosphate remained trapped upon the histidyl. Thus in the absence of a protonated amino acid N-terminal no catalysis of hydrolysis was observed. To further assess the importance of the cationic N-terminus, we performed the reaction with hercynine -a histidine with an Nterminal -NMe3 + group. Here, we observed again the accelerated disappearance of imidazole phosphate but orthophosphate formation was not accelerated compared to the background (Figure , Ã trace). The absence of catalysis in the presence of a cationic trimethylated N-terminal suggests that the hydrolysis transition state may be stabilised by the formation of an intramolecular H-bond between the cationic ammonium N-terminal and the departing phosphate group. This is in addition to any stability conferred via favourable electrostatic interactions between the two groups. Therefore the combination of the imidazolyl ring and the N-terminal ammonium are required to catalyse the reaction.