Capturing Unknown Substrates via in Situ Formation of Tightly Bound Bisubstrate Adducts: S-Adenosyl-vinthionine as a Functional Probe for AdoMet-Dependent Methyltransferases

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• 作者：Wanlu Qu ; Kalli C. Catcott ; Kun Zhang ; Shanshan Liu ; Jason J. Guo ; Jisheng Ma ; Michael Pablo ; James Glick ; Yuan Xiu ; Nathaniel Kenton ; Xiaoyu Ma ; Richard I. Duclos Jr. ; Zhaohui Sunny Zhou
• 刊名：Journal of the American Chemical Society
• 出版年：2016
• 出版时间：March 9, 2016
• 年：2016
• 卷：138
• 期：9
• 页码：2877-2880
• 全文大小：343K
• ISSN：1520-5126

文摘

Identifying an enzyme’s substrates is essential to understand its function, yet it remains challenging. A fundamental impediment is the transient interactions between an enzyme and its substrates. In contrast, tight binding is often observed for multisubstrate-adduct inhibitors due to synergistic interactions. Extending this venerable concept to enzyme-catalyzed in situ adduct formation, unknown substrates were affinity-captured by an S-adenosyl-methionine (AdoMet, SAM)-dependent methyltransferase (MTase). Specifically, the electrophilic methyl sulfonium (alkyl donor) in AdoMet is replaced with a vinyl sulfonium (Michael acceptor) in S-adenosyl-vinthionine (AdoVin). Via an addition reaction, AdoVin and the nucleophilic substrate form a covalent bisubstrate-adduct tightly complexed with thiopurine MTase (2.1.1.67). As such, an unknown substrate was readily identified from crude cell lysates. Moreover, this approach is applicable to other systems, even if the enzyme is unknown.