To better understand why mutant M45F also displayed a tremendous increase in activity compared to HheG wild type, kinetic parameters of this variant in the azidolysis of cyclohexene oxide (1) were determined using our BTB assay (Table ). This revealed not only a 5 to 15-fold increase in maximal reaction velocity (kobs,max) of mutant M45F (rate improvement varies when either the kinetics for epoxide or azide are considered), but also a significant improvement in azide binding compared to HheG wild type. Interestingly, this loop mutation seems to enhance the cooperativity for azide binding, as the respective Hill coefficient n increased as well. A recent conformational landscape analysis of HheG demonstrated that loop3947 would affect the presence and shape of substrate tunnel T3 in HheG. The latter might impact nucleophile binding in HheG as well.