AI Article Synopsis
- * Researchers extended the concept of tight binding in multisubstrate-adduct inhibitors to discover unknown substrates using a modified methyltransferase system.
- * The process involved replacing a component of S-adenosyl-methionine with S-adenosyl-vinthionine, allowing for the identification of unknown substrates from crude cell lysates and applicable to different systems.
Article Abstract
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.
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| http://dx.doi.org/10.1021/jacs.5b05950 | DOI Listing |
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