To unveil if the observed activity increase of HheC mutant F12Y is induced by an improved substrate binding or rather a significantly elevated reaction rate, we measured kinetic data for the dehalogenation of haloalcohol 1f and the ring opening of epoxide 1 with azide using the halide release assay as well as our BTBbased pH assay, respectively. Interestingly, this kinetic analysis revealed that the F12Y mutation only slightly impacted KM or K50 for the substrates 1f and 1 (Table ). In contrast, the maximal reaction rates were considerably increased -3.5-fold for the dehalogenation of 1f and at least 5-fold in the azidolysis of 1 (rate improvement varies when either the kinetics for epoxide or azide are considered). Thus, the improved performance of HheC F12Y in dehalogenation and epoxide ring opening is solely caused by an enhancement of reaction velocities. For comparison, the corresponding K50 values of HheC mutants T7S and G14A for binding of epoxide 1 were increased by a factor of 2 or more compared to the wild type and mutant F12Y, indicating a lower substrate affinity of those motif 1 mutants in HheC. Moreover, HheC T7S exhibited an at least two-fold higher kobs,max compared to wild type, while the respective kobs,max of HheC G14A in the azidolysis of 1 was drastically reduced, both in line with our determined specific activities. Unfortunately, true kcat values could not be determined in this epoxide ring opening reaction due to the high K50 values of all HheC mutants towards azide and the strong chemical background azidolysis of 1 occurring at azide concentrations above 100 mM. Therefore, kinetic measurements with varied epoxide concentration were performed at non-saturating azide concentration, yielding significantly lower kobs,max values compared to kinetic measurements with fixed epoxide and varying azide concentrations (Table ). Importantly, all three HheC mutants do also exhibit higher K50 values as well as a considerably stronger cooperativity in azide bindingbased on higher Hill coefficients nHthan the corresponding wild-type enzyme (Table ). Thus, those conserved residues in sequence motif 1 of HheC are indeed influencing nucleophile binding to a great extent. In contrast, changes in the kinetic parameters of HheG mutants compared to wild type in the azidolysis of epoxide 3 are less dramatic (Table ), which is again in agreement with respective specific activities of HheG mutants determined for this reaction. The observed cooperativity for azide binding, however, again varies significantly depending on the introduced mutation. Table . Kinetic parameters of selected HheC mutants in the dehalogenation of 1,3-dichloro-2-propanol (1f) (determined by halide release assay) as well as the azidolysis of epichlorohydrin (1) (determined by our BTB assay). For the latter reaction, first the epoxide concentration was varied while keeping the azide concentration constant at 100 mM; afterwards the azide concentration was varied fixing the epoxide concentration at 100 mM. The Michaelis-Menten equation was used to fit the resulting data for the dehalogenation of 1f, whereas the Hill equation was used for fitting the experimental data obtained for the ring opening of 1 with azide. Next, we probed the enantioselectivity of our motif 1 mutants in epoxide ring opening reactions with azide to test if motif 1 mutations affected also the selectivity of these enzymes. For variants of HheC, we selected the terminal epoxides 1 and 3 as substrates, while we used 3 and the cyclic epoxide 2 for HheG variants. Our choice of epoxide 3 was primarily motivated by the opposite regioselectivity of HheC and HheG in their ring opening of this substrate (Figure ). The non-catalyzed reaction preferentially yields 2-azido-2-phenylethan-1-ol (3a.1) through nucleophilic attack at the benzylic α-carbon. While HheG enforces this inherent preference, HheC exhibits selectivity for attack at the terminal β-position. Biotransformations analyzed by chiral GC revealed that most of the motif 1 mutations in HheC decreased enantioselectivity significantly compared to the wild-type enzyme, independently of the epoxide substrate (Table ). In contrast, mutants T7S and F12Y displayed a greatly increased enantioselectivity in the conversion of 1 with azide, while the extremely high enantioselectivity of the wild type enzyme with 7 was maintained. This indicates a considerable impact of motif 1 residues on the enantioselectivity of HheC. On the other hand, the enantioselectivity of the studied HheG mutants in the ring opening of 2 and 3 hardly changed compared to HheG wild type (Table ). For comparison, mutations at the central aromatic residue Y18 in the homologous HheG enzyme from Acidimicrobiia bacterium did affect enantioselectivity in the ring opening of 3 with cyanate. Apart from this overall varying influence of motif 1 residues on enantioselectivity, all herein studied HheC and HheG mutants retained the wild-type enantiopreference for the conversion of (S)-1 and (R)-3. Following these activity and enantioselectivity studies, we also examined the thermal stability of active motif 1 mutants of HheC and HheG by differential scanning fluorimetry (also known as thermofluor assay), as a positive impact of mutation F12Y on the thermostability of HheC has previously been reported. Our analysis revealed a considerable stabilizing effect for mutations F12H (+10.5 K), F12Y (+10.1 K) and G14A (+7.3 K) in HheC (Figure ). The latter is especially surprising as this mutation heavily decreased enzyme activity. In contrast, the opposite trend in thermal stability has previously been reported for the exchange of the equivalent glycine residue in SDR motif 1 of Drosophila alcohol dehydrogenase, and was also observed in our study for the corresponding mutation G20A in HheG. The herein reported stabilizing effect of mutation F12Y in HheC was previously attributed to the formation of an additional hydrogen bond with residue T131 compared to wild-type HheC, which we could confirm based on our computational results (see below). A slight increase (+2.8 K) in the apparent melting temperature of HheG upon exchange of the corresponding tyrosine 18 by phenylalanine becomes apparent from Figure as well. a Reactions were carried out in a total volume of 1 mL in 50 mM Tris•SO4, pH 7.0, at 30 °C (HheC) or 22 °C (HheG) and 900 rpm using 10-400 µg mL -1 purified enzyme. Samples were taken after 15 min (epoxide 1) or 1 h (epoxide 3), extracted with an equal volume of tert-butyl methyl ether and analyzed by achiral and chiral GC. b Conversion and enantioselectivity towards formation of product 2-azido-1-phenylethan-1-ol (3a.2) through nucleophilic attack at the terminal β-position. c In the non-catalyzed chemical background reaction, formation of product 2-azido-2-phenylethan-1-ol (3a.1) through nucleophilic attack at the benzylic α-carbon is preferred. d Conversion and enantioselectivity towards formation of product 2-azido-2-phenylethan-1-ol (3a.1) through nucleophilic attack at the benzylic α-carbon.