Reaction of cis-3-Chloroacrylic Acid Dehalogenase with an Allene Substrate, 2,3-Butadienoate: Hydration via an Enamine

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• 作者：Gottfried K. Schroeder ; William H. Johnson ; Jr. ; Jamison P. Huddleston ; Hector Serrano ; Kenneth A. Johnson ; Christian P. Whitman
• 刊名：The Journal of the American Chemical Society
• 出版年：2012
• 出版时间：January 11, 2012
• 年：2012
• 卷：134
• 期：1
• 页码：293-304
• 全文大小：602K
• 年卷期：v.134,no.1(January 11, 2012)
• ISSN：1520-5126

文摘

cis-3-Chloroacrylic acid dehalogenase (cis-CaaD) catalyzes the hydrolytic dehalogenation of cis-3-haloacrylates to yield malonate semialdehyde. The enzyme processes other substrates including an allene (2,3-butadienoate) to produce acetoacetate. In the course of a stereochemical analysis of the cis-CaaD-catalyzed reaction using this allene, the enzyme was unexpectedly inactivated in the presence of NaBH₄ by the reduction of a covalent enzyme鈥搒ubstrate bond. Covalent modification was surprising because the accumulated evidence for cis-CaaD dehalogenation favored a mechanism involving direct substrate hydration mediated by Pro-1. However, the results of subsequent mechanistic, pre-steady state and full progress kinetic experiments are consistent with a mechanism in which an enamine forms between Pro-1 and the allene. Hydrolysis of the enamine or an imine tautomer produces acetoacetate. Reduction of the imine species is likely responsible for the observed enzyme inactivation. This is the first reported observation of a tautomerase superfamily member functioning by covalent catalysis. The results may suggest that some fraction of the cis-CaaD-catalyzed dehalogenation of cis-3-haloacrylates also proceeds by covalent catalysis.