Effects of Temperature on the Kinetic Isotope Effects for Proton and Hydride Transfers in the Active Site Variant of Choline Oxidase Ser101Ala
نویسندگان
چکیده
Choline oxidase catalyzes the oxidation of choline to glycine betaine. The reaction includes betaine aldehyde as an intermediate. FAD is reduced by the alcohol substrate, betaine aldehyde intermediate and oxidized by molecular oxygen to give hydrogen peroxide. In this study, the Ser101Ala variant of choline oxidase was prepared to elucidate the contribution of the hydroxyl group of Ser101 in the proton and hydride transfer reactions for proper preorganization and reorganization of the active site towards quantum mechanical tunneling. The thermodynamic parameters associated with the enzyme-catalyzed OH and CH bond cleavages and the temperature dependence of the associated solvent and substrate kinetic isotope effects were investigated using a stopped-flow spectrophotometer. The proton and hydride transfer have been shown to be occurring via quantum tunneling in CHO-S101A enzyme. INDEX WORDS: Choline oxidase, Quantum tunneling, Proton transfer, Hydride transfer, Kinetic complexity EFFECTS OF TEMPERATURE ON THE KINETIC ISOTOPE EFFECTS FOR PROTON AND HYDRIDE TRANSFERS IN THE ACTIVE SITE VARIANT OF CHOLINE OXIDASE
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