Structural effect of amine N-oxides on the facilitation of α-glucosidase-catalyzed hydrolysis reactions.

Activation and stabilization of enzymes is an important issue in their industrial application. We recently reported that synthetic betaines, derived from cellular metabolites, structure-dependently increased the activity and stability of various enzymes including hydrolases, oxidases, and synthetases simply by mixing them into the reaction buffer. In this report, we focus on amine N-oxides, which are similarly important metabolites in cells with a highly polarized N-oxide bond, and investigate their enzyme stabilization and activation behavior. It was revealed that synthetic amine N-oxides structure-dependently activate α-glucosidase-catalyzed hydrolysis reactions similarly to betaines. The subsequent comparison of the kinetic parameters, the optimal concentration range for activation, and the maximal activity, suggested that amine N-oxides facilitate hydrolysis reactions via the same mechanism as betaines, because no differences were confirmed. However, the enzyme stabilization effect of amine N-oxides was slightly superior to that of betaines and the temporal stability of the enzyme in aqueous solutions was higher in the low amine N-oxide concentration range. The rheological properties, CD spectra, and dynamic fluorescence quenching experiments suggested that the suppression of unfavorable conformational perturbation was related to the difference in the hydration environments provided by the surrounding water molecules. Thus, we clarified that amine N-oxides facilitate enzyme reactions as a result of their similarity to betaines and provide a superior stabilizing effect for enzymes. Amine N-oxides show potential for application in enzyme storage and long-term reactions.

Significance of anionic functional group in betaine-type metabolite analogs on the facilitation of enzyme reactions.

Using synthetic sulfobetaine library, the enzyme activation behavior has been investigated. Comparison of enzyme activation behavior revealed that sulfobetaines equally facilitate enzyme reactions, being consistent with that of carboxybetaines. The subsequent kinetic and solution property analyses clarified that both the kinetic parameter and hydration property changes are identical with those of carboxybetaines, indicating that the difference in the anionic functional group of the betaine structure scarcely affects the enzyme activation. On the other hand, comparison of carboxy- or sulfo-betaines with tetraalkylammonium salts, whose counteranion binds to the ammonium cation intermolecularly, revealed that the activation ability for enzymes of tetraalkylammonium salts is considerably smaller than that of carboxy- or sulfo-betaines. These findings give us a hint to design the useful betaine-type enzyme activators.