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

• Published in: Chembiochem : a European journal of chemical biology 2017-04, Vol.18 (7), p.613-617
• Main Authors: Catcott, Kalli C., Yan, Jing, Qu, Wanlu, Wysocki, Vicki H., Zhou, Zhaohui Sunny
• Format: Article
• Language: English
• Subjects: bioorganic chemistry; Carbon Radioisotopes; Enzymes; Escherichia coli - metabolism; Isotope Labeling; Mass Spectrometry; Methyltransferases - chemistry; Methyltransferases - metabolism; Molecular Probes - analysis; Molecular Probes - chemistry; Nitrogen Radioisotopes; S-Adenosylhomocysteine - analogs & derivatives; S-Adenosylhomocysteine - chemistry; S-Adenosylhomocysteine - metabolism; S-Adenosylmethionine - chemistry; S-Adenosylmethionine - metabolism; Scientific imaging; substrate identification; Substrate Specificity; transferases
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.201600634

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. Feeling IsoLAITed: Isotope‐labeled, activity‐based identification and tracking (IsoLAIT) is a framework designed to identify enzyme–substrate pairs from complex matrices. By replacing a common enzyme substrate with an isotope‐labeled, activity‐based probe, detection of the [enzyme⋅substrate⋅probe] complex by native mass spectrometry is rendered facile, even if the components are of unknown chemical nature and mass.