期刊
CHEMBIOCHEM
卷 18, 期 7, 页码 613-617出版社
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cbic.201600634
关键词
bioorganic chemistry; enzymes; mass spectrometry; substrate identification; transferases
资金
- National Science Foundation [DBI-0923551, DBI-1455654]
- National Institute of General Medical Sciences (NIGMS) at the National Institutes of Health [R01GM101396]
The enzyme-substrate complex is inherently transient, rendering its detection difficult. In our framework designed for bisubstrate systemsisotope-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 [enzymesubstrateprobe] 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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