Having confirmed that an N-terminal phosphoramidate is not the catalytic species and thus the catalysis must arise from the imidazolyl ring on histidine (catalysis of phosphate ester hydrolysis by imidazolyl rings are known in the literature ) we next sought to see if catalysis by the imidazolyl ring acted alone or in concert with the ammonium at the N-terminus. We performed the hydrolysis reaction in the presence of 50 mM acetylated N-terminal histidine (Figure and and).