We further characterized the Michaelis-Menten kinetics of two intermediate variants ACCO I184A K158I F91L K172Y, ACCO I184A K158I F91L K172Y K93Q and the final variant ACCOCHF using purified enzymes in the presence of stoichiometric quantities of dithionite as the reductant (Figs. ). Michaelis-Menten kinetics showed that while the kcat value of these ACCO variants remained approximately unchanged (2.1 min -1 , 2.5 min -1 and 2.3 min -1 , respectively), the KM value of this evolutionary series decreased from 520 µM to 420 µM to 210 µM. Together, the kcat/KM value of these variants increased from 4.0 mM -1 min -1 to 6.0 mM -1 min -1 to 11 mM -1 min -1 . Thus, these results indicated that the improved enzymatic towards C-H fluorination originated from enhanced substrate binding, which may arise from substrate tunnel engineering.