The histidyl catalysed phosphate transfer proceeded again via the formation of a phosphorylated imidazolyl histidyl intermediate. This intermediate was observed in the 31 P NMR spectra (Figure ) and formed typically in a 30 -35 % yield after 16 h. Unlike in the hydrolysis reactions above, we did observe formation of a minor amount (typically in a < 5 % yield after 16 h) of N-terminal histidyl phosphoramidate (Figure .62 ppm). Given that, in all cases, the yield of the phosphorylated imidazolyl intermediate significantly exceeded that of the Nterminal phosphoramidates and our above results on hydrolysis, we consider that the phosphorylated imidazolyl intermediate is the most catalytically relevant species. The results from the wetto-dry experiments displayed good repeatability in the yield of glycerol phosphate at each time point. A histogram (SI Figure ) of the standard deviations from all data points in Figure shows that the standard deviation was typically < 2.5 %.