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  • br Discussion The differences in the kinetic properties

    2019-11-08


    Discussion The differences in the kinetic properties of L-AmDH and LeuDH presented herein reflect a different kinetic mechanism. When compared to its parent enzyme, L-AmDH displayed a different substrate binding order, lower affinity for the keto substrate, and stronger product inhibition. The two key residues which must be mutated to produce an AmDH from an amino the original source dehydrogenase (K68 and N261 for L-AmDH) form hydrogen bonds with the acid group on the substrate [1]. The mutations to serine and leucine, respectively, permit the binding of methyl ketones but with lower affinity compared to keto acids (Table 3). The second-order rate constants for pentanone and 2-aminopentane are both three orders of magnitude lower than those for leucine and ketoleucine. Additionally, solvent viscosity effects demonstrated that an isomerization of the enzyme-substrate complex, important for LeuDH catalysis, was not observed for L-AmDH, likely owing to a change in rate-limiting step from LeuDH to L-AmDH functionality. Poor NH3 binding was previously hypothesized to cause the 30-fold decrease in activity between L-AmDH and LeuDH [19]. However, at reaction conditions relevant for large-scale synthesis, i.e. high concentrations of all substrates and pH value of 8.5, the KM,NH4 values for LeuDH and L-AmDH are much closer than had been expected based on reports at pH ∼ 9.6, where KM,NH4 values of < 300 mM were reported for LeuDHs [20], [21], [22]. Since the calculated KM,NH4 value for each enzyme was near the solubility limit of NH4Cl of ∼ 6 M, saturating conditions for NH4Cl substrate could not be realized. The large KM,NH4 values paired with the lack of a constant kinetic term for LeuDH or L-AmDH suggests that ammonia in neutral or ionic form (NH3 or NH4+) does not bind to the enzyme at all, but rather that free ammonia attacks the bound ketone or keto-acid directly, as previously proposed for PheDH [23] and GluDH [24]. The pH value of 8.5 was chosen for the present study to match the pH value for large-scale synthesis, where a cofactor regeneration enzyme must be employed to economize cofactor use and to drive high conversion. The most common of these regeneration enzymes are formate dehydrogenase (FDH), with a pH optimum around pH 7.5 [25], and glucose dehydrogenase (GDH), with a pH optimum of 8.5 [26]. Additionally, L-AmDH and FDH are destabilized at higher pH values (unpublished data). Both L-AmDH and LeuDH have lower activity at pH 8.5 than at pH 9.6, the most common pH value for conversions with AmDHs [10], [13], [27]. The pH optima of ketoleucine amination and leucine deamination for LeuDH are at pH 9.5 and pH 11, respectively [28]. The pKa value for ammonium at ionic strength of 4 M is around pH 9.7 [29], [30], so at pH 8.5 the protonated form (NH4+) dominates, which contributes to the high values of KM,NH4. A standard ionic strength of 4 M, (equal to the maximum NH4Cl concentration used), was maintained for both amination and deamination, as a change of 1.5 M ionic strength can double/half the KM,keto value (see Fig. S.3 in the Supplemental Information). The results for the kinetic mechanism of LeuDH are consistent with previously reported data, except for the observed competitive inhibition by leucine on ketoleucine (Table 4). For reductive amination, there is wide agreement for a strictly ordered sequential mechanism of substrate binding, though the exact order has not been consistent. There is broad agreement that NAD(P)H binds first, supported by the observation of competitive inhibition between NAD(P)+ and NAD(P)H, indicating both forms of the cofactor can bind to free enzyme. For LeuDH [31], [20], [21], [22], AlaDH [32], [33], and PheDH [14], [34], [35], previous studies have indicated that ammonia binds either second or third. Other than the lack of the constant term in the reductive amination direction (Table 1), the fitted rate laws for LeuDH in both directions are consistent with an ordered sequential mechanism in which NADH binds first, followed by ketoleucine, and then ammonia. This binding order is consistent with the lack of a [B] term in the denominator of the rate equation, i.e. absence of an enzyme-ketoleucine complex, and has been proposed for other leucine dehydrogenases [22], [31].