Further Insight into the Mechanism of Stereoselective Proton Abstraction by Bacterial Copper Amine Oxidase

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• 作者：Masayasu Taki ; Takeshi Murakawa ; Takuya Nakamoto ; Mayumi Uchida ; Hideyuki Hayashi ; Katsuyuki Tanizawa ; Yukio Yamamoto ; Toshihide Okajima
• 刊名：Biochemistry
• 出版年：2008
• 出版时间：July 22, 2008
• 年：2008
• 卷：47
• 期：29
• 页码：7726 - 7733
• 全文大小：2976K
• 年卷期：v.47,no.29(July 22, 2008)
• ISSN：1520-4995

文摘

During the catalytic reaction of copper amine oxidase, one of the two prochiral hydrogen atoms at the C1 position of substrate amine is stereoselectively abstracted by a conserved Asp residue serving as a general base. Using stereospecifically deuterium-labeled enantiomers of 2-phenylethylamine, we previously showed that the pro-S α-proton is abstracted by the enzyme from Arthrobacter globiformis (AGAO) [Uchida, M., et al. (2003) Biosci. Biotechnol. Biochem. 67, 2664−2667]. More recently, we have also demonstrated that the pro-S selectivity of α-proton abstraction is fully retained even in the reaction of a mutant AGAO lacking the catalytic base [Chiu, Y.-C., et al. (2006) Biochemistry 45, 4105−4120]. On the basis of these findings, we have proposed that the stereoselectivity of α-proton abstraction is primarily determined by the conformation of the Schiff base intermediate formed between the substrate and the topa quinone cofactor (TPQ), stabilized by the binding of the distal part of the substrate to a hydrophobic pocket of the enzyme. In this conformation, the pro-S hydrogen atom to be abstracted is nearly perpendicular to the plane of the Schiff base−TPQ conjugate system, achieving the maximum overlap of σ- and π-orbitals. To further elucidate the stereochemical details, we have synthesized stereospecifically deuterium-labeled enantiomers of ethylamine, a very poor substrate for AGAO, in addition to those structurally related to the preferred substrate, 2-phenylethylamine. In marked contrast to the nearly complete pro-S selectivity of α-proton abstraction for most substrates that have been examined, the stereoselectivity for ethylamine decreased significantly to as little as 88%. The crystal structure of AGAO soaked with ethylamine showed very poor electron densities for the substrate Schiff base intermediate, showing that its conformation is not defined uniquely. Thus, the stereoselectivity of α-proton abstraction during the copper amine oxidase reaction is closely associated with the conformational flexibility of the substrate Schiff base intermediate.