Living organisms accumulate several metabolites in the cell to survive in harsh environments, where concentration of the metabolites sometimes increases up to several mol/L. To understand why living organisms select the metabolites among thousands of cellular metabolites, we synthesized a structurally diverse metabolite analog library derived from a naturally occurring metabolite, glycine betaine, and investigated the influence of the chemical structure on the activity and stability of biomolecules. To date, we have found clarified the significance of the chemical structure on the facilitation of hydrolases-mediated hydrolysis reactions. In particular, the analog possess triplicate n-butyl chains in ammonium cation of glycine betaine shows the highest activation of hydrolysis reaction. Furthermore, the activation can be observed for increment of not only hydrolysis rate but also substrate specificity, thermal stability and salt tolerance. In this presentation, we would like to report the activation mechanism by analyzing the solution property of metabolite analogs. According to our previous studies, the analogs similarly facilitate various enzyme reactions by dissolving them into buffer solution at the same concentration, suggesting that the activation is indirect manner. Solution property of the analog solution was analyzed by dynamic light scattering, vapor pressure osmolarity, dielectric constant and water density measurements. Although the analogs possess hydrophobic aliphatic chains, they dispersed in aqueous media as a monomer. They were solvated by plenty of water molecules, number of which was far large in comparison to that for hydrophilic glucose. In addition, the water density of hydration layer is low. Such large difference in hydration layer lead to induce a phase separation of the molecules, suggesting that hydrophilic enzymes and substrates are excluded from analogs which results in increase in the collision efficacy. It is supported by the kinetic parameter changes (decrease in K<sub>m</sub>). Besides, large osmolarity change induces the decrement of water activity, which should change the solvation environments of enzymes. That slightly suppresses the conformational perturbation of enzymes, resulting in increase the thermal stability and substrate specificity of enzymes.

Mechanistic study of unusual enzyme activation caused by addition of inert betaine-type metabolite and the analogs

249th National Meeting (2015)

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