We assessed in this system the importance of activating and trapping orthophosphate in the KSTA imidazole phosphate prior to the phosphorylation of the histidyl catalyst. It is conceivable that the histidyl peptides could be directly phosphorylated by carbamoyl phosphate and we wished to explore how this affected the phosphorylation of glycerol. We performed the experiment under identical conditions with 100 mM histidine but without imidazole present (Figure , tan bar, SI Section S4.10). Over the three cycles, the yields of phosphorylated glycerol were ~4-fold lower per cycle than for the reactions where both histidine and imidazole are present (Figure , tan vs. red bars). The yields from this histidine-only reaction were lower even than the uncatalysed reaction (Figure , tan vs. magenta bars). This result demonstrates the importance of the phosphorylation pathway proceeding via the formation of the KSTA imidazole phosphate rather than proceeding directly through the catalyst.