Our results show that ParaCDPS generated more product using His tRNAHis + Phe-DBE, in disagreement to what would be expected if histidine was occupying P1 if the hypothesis above was correct. Consequently, we investigated both enzymes using a trapped acyl-enzyme intermediate experiment developed in-house, which exploits the minimal aa-DBE substrate as a chemical probe to verify substrate binding order. Here we saw that ParaCDPS accepts Phe in P1 instead of His whereas ParcuCDPS accepts His in P1 (Fig. ). This therefore corroborates our hypothesis, and most likely DBE-aa occupying P2 can sample several unproductive conformations leading to DBE hydrolysis before successful product formation.