We explored how the structure of the histidyl peptide catalyst affected its ability to catalyse the phosphate transfer reaction. We performed the reaction with a range of different histidyl peptides which had histidyl residues at either the N-terminus, in the centre of the peptide chain or at the C-terminus (Figure , SI Section S3.2 -S3. 16). In all cases, the phosphorylation was catalysed but the catalytic activity varied depending upon the position of the histidyl residue in the peptide chain (Figure ). The general trend in catalytic performance was that N-terminus > centre > C-terminus histidyls. The closer the histidyl is in proximity to the cationic ammonium N-terminal the greater the acceleration in the phosphate transfer reaction. This is consistent with the results from the hydrolysis experiments above. In these reactions the amino acid histidine proved to be the most effective catalyst for transferring phosphate to glycerol.