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.

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.

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The cover picture shows a new approach to identifying enzyme–substrate pairs. The top panel shows a typical catalytic cycle of an enzyme, in which the enzyme binds the substrates, converts them to products, and releases them. Due to their inherently transient nature, with few exceptions, the weakly bound enzyme–substrate complexes cannot be directly observed by mass spectrometry. The bottom panel shows our new approach dubbed IsoLAIT: isotope‐labeled, activity‐based identification and tracking. IsoLAIT uses a substrate analogue as a probe. The enzyme catalyzes bisubstrate–adduct formation between the probe and the enzyme's native substrate. The resulting tightly bound [enzyme⋅substrate⋅probe] complex can be analyzed by native mass spectrometry; and each individual component can be further interrogated by tandem mass spectrometry. The probe's isotopic pattern is telltale sign for such complexes; therefore, even without a priori knowledge of their chemical nature, unknown components can be identified.