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Identifying Unknown Enzyme-Substrate Pairs from the Cellular Milieu with Native Mass Spectrometry.

ABSTRACT: The enzyme-substrate complex is inherently transient, rendering its detection difficult. In our framework designed for bisubstrate systems-isotope-labeled, activity-based identification and tracking (IsoLAIT)-the common substrate, such as S-adenosyl-l-methionine (AdoMet) for methyltransferases, is replaced by an analogue (e.g., S-adenosyl-l-vinthionine) that, as a probe, creates a tightly bound [enzyme?substrate?probe] complex upon catalysis by thiopurine-S-methyltransferase (TPMT, EC 2.1.1.67). This persistent complex is then identified by native mass spectrometry from the cellular milieu without separation. Furthermore, the probe's isotope pattern flags even unknown substrates and enzymes. IsoLAIT is broadly applicable for other enzyme systems, particularly those catalyzing group transfer and with multiple substrates, such as glycosyltransferases and kinases.

SUBMITTER: Catcott KC

PROVIDER: S-EPMC6483605 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Identifying Unknown Enzyme-Substrate Pairs from the Cellular Milieu with Native Mass Spectrometry.

Catcott Kalli C KC Yan Jing J Qu Wanlu W Wysocki Vicki H VH Zhou Zhaohui Sunny ZS

Chembiochem : a European journal of chemical biology 20170314 7

The enzyme-substrate complex is inherently transient, rendering its detection difficult. In our framework designed for bisubstrate systems-isotope-labeled, activity-based identification and tracking (IsoLAIT)-the common substrate, such as S-adenosyl-l-methionine (AdoMet) for methyltransferases, is replaced by an analogue (e.g., S-adenosyl-l-vinthionine) that, as a probe, creates a tightly bound [enzyme⋅substrate⋅probe] complex upon catalysis by thiopurine-S-methyltransferase (TPMT, EC 2.1.1.67). ...[more]

PMID: 28140508

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