In the case of 100 mM histidine, we presume that the absence of catalysed phosphate transfer in the first cycle is due to the balance in the reaction rates which favours histidine catalysing the hydrolysis of imidazole phosphate (histidine was the most effective catalyst for the hydrolysis of imidazole phosphate (Figure )). In the second cycle, catalysis of glycerol phosphate formation was observed with 100 mM histidine. We believe this is a result of the outcome of the first cycle influencing the second cycle. At the end of the first cycle there remains imidazole phosphate and phosphorylated histidine in the paste. The presence of these species in combination with the refuelling of the reaction by cyanate pushes the reaction quotients towards formation of imidazole phosphate and phosphorylated histidine. This is seen in the higher yield at the end of the second cycle compared to the first cycle of imidazole phosphate (2 nd cycle = 26.9 ± 0.6 % vs. 1 st cycle = 14.3 ± 0.3 %) and phosphorylated histidine (2 nd cycle = 16.1 ± 1.8 % vs. 1 st cycle = 7.1 ± 0.6 %) (SI Figures and). This leads ultimately to the observed increased yield of phosphorylated glycerol in the second and third cycles with 100 mM histidine.