Effect of a conservative mutation of an active site residue involved in substrate binding on the hydride tunneling reaction catalyzed by choline oxidase

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• 作者：Osbourne Quaye ; Giovanni Gadda
• 关键词：Choline oxidase ; Flavoprotein oxidase ; Hydride transfer ; Hydride tunneling ; Kinetic isotope effects ; Temperature effects ; Flavoprotein
• 刊名：Archives of Biochemistry and Biophysics
• 出版年：2009
• 出版时间：September 2009
• 年：2009
• 卷：489
• 期：1-2
• 页码：10-14
• 全文大小：316 K

文摘

The reaction of alcohol oxidation catalyzed by choline oxidase was previously shown to occur through the tunneling of a hydride ion from the α-carbon of an activated alkoxide species to the N(5) atom of FAD within a highly preorganized enzyme-substrate complex [G. Gadda, Biochemistry 47 (2008) 13745–13753]. In the present study, a glutamate residue (312) that participates in substrate binding has been replaced with aspartate by site-directed mutagenesis, and the effect of the substitution on the kinetic parameters of the enzyme and the mechanism of hydride ion transfer were investigated using stopped-flow spectrophotometry. The thermodynamic parameters associated with the enzymatic reaction catalyzed by the mutant enzyme and the temperature dependence of the kinetic isotope effects are consistent with a hydride transfer reaction occurring either through environmentally assisted tunneling or a classical over-the-barrier transition state, but not within a preorganized enzyme-substrate complex. In contrast, less than threefold changes in the K_d values for choline were observed in the mutant enzyme with respect to the wild-type enzyme. Thus, the conservative substitution of an active site residue that is involved in substrate binding, but not directly in catalysis, affects primarily the k_red value that reports on catalysis and minimally the K_d value that reports on binding, thereby providing an example of the interplay that these kinetic parameters may have in enzymatic reactions.