Having observed the catalysed hydrolysis of imidazole phosphate we next sought to confirm that the phosphorylated histidyl was the catalytic species in the reaction. In principle, the catalysis could be via either the formation of a phosphorylated imidazolyl histidine (observed in 31 P NMR spectra) or via an Nterminal phosphoramidate (not observed in our P NMR spectra, expected chemical shift of d ~ 5 -6 ppm at pH 7.5). To determine if the catalysis proceeded via an N-terminal phosphoramidate we performed the hydrolysis reaction in the presence of 50 mM alanine (Figure , p and r traces). No catalysis of the hydrolysis reaction was observed and the disappearance of imidazole phosphate and the appearance of orthophosphate overlaid well with the uncatalysed reaction. Admittedly, the formation of the N-terminal phosphoramidate is unlikely, as at pH 7.5 the N-terminus is found as the ammonium species and thus the lone pair on nitrogen is unavailable to act as a nucleophile.