Identification of the Catalytic Base for Alcohol Activation in Choline Oxidase

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• 作者：Crystal Smitherman ; Kunchala Rungsrisuriyachai ; Markus W. Germann ; Giovanni Gadda
• 刊名：Biochemistry
• 出版年：2015
• 出版时间：January 20, 2015
• 年：2015
• 卷：54
• 期：2
• 页码：413-421
• 全文大小：410K
• ISSN：1520-4995

文摘

Choline oxidase catalyzes the oxidation of choline to glycine betaine through a two-step, four-electron reaction with betaine aldehyde as an intermediate. Oxygen is the final electron acceptor. Alcohol oxidation is initiated by the removal of the substrate hydroxyl proton by an unknown active site residue with a pK_a value of 鈭?.5. In the crystal structure of the enzyme in complex with glycine betaine, H466 is 鈮?.1 脜 from the carboxylate oxygen of the reaction product, suggesting a possible role in the proton abstraction reaction catalyzed by the enzyme. H466, along with another potential candidate, H351, was previously mutated to alanine, but this failed to establish if either residue was involved in activation of the substrate. In this study, single variants of choline oxidase with H466 and H351 substituted with glutamine were prepared, purified, and characterized. The k_cat and k_cat/K_m values of the H351Q enzyme in atmospheric oxygen were 45- and 5000-fold lower than those of the wild-type enzyme, respectively, whereas the H466Q enzyme was inactive when assayed polarographically with choline. In the H466Q enzyme, the rate constant for anaerobic flavin reduction (k_red) with choline was 1 million-fold lower than in the wild-type enzyme. A comparison of the fluorescence, circular dichroism, and ¹H nuclear magnetic resonance spectroscopic properties of the H466Q enzyme and the wild-type enzyme is consistent with the mutation not affecting the topology of the active site or the overall fold of the protein. Thus, the change in the k_red value and the lack of oxygen consumption upon mutation of histidine to glutamine are not due to misfolded protein but rather to the variant enzyme being unable to catalyze substrate oxidation. On the basis of the kinetic and spectroscopic results presented here and the recent structural information, we propose that H466 is the residue that activates choline to the alkoxide for the subsequent hydride transfer reaction to the enzyme-bound flavin.