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Observation of a chemically labile, noncovalent enzyme intermediate in the reaction of metal‐dependent Aquifex pyrophilus KDO8PS by time‐resolved mass spectrometry
Author(s) -
Roberts Anne,
Furdui Cristina,
Anderson Karen S.
Publication year - 2010
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.4594
Subject(s) - chemistry , mass spectrometry , electrospray ionization , reaction intermediate , non covalent interactions , metal , electrospray , molecule , organic chemistry , chromatography , catalysis , hydrogen bond
The direct detection of intermediates in enzymatic reactions can yield important mechanistic insights but may be difficult due to short intermediate lifetimes and chemical instability. Using a rapid‐mixing device coupled with electrospray ionization time‐of‐flight mass spectrometry, the noncovalent hemiketal intermediate in the reaction of metal‐dependent 3‐deoxy‐D‐manno‐octulosonate‐8‐phosphate (KDO8P) synthase from Aquifex pyrophilus was observed in the millisecond time range. Using single turnover conditions, the noncovalent complexes of enzyme with Cd 2+ :phosphoenolpyruvate, Cd 2+ :phosphate, Cd 2+ :KDO8P, and Cd 2+ :intermediate complexes were resolved. The intermediate complex is present during times ranging from 50–630 ms, indicating that the intermediate builds up at the ambient temperatures of the experiment. This represents the first direct detection of the intermediate with a native metal‐dependent KDO8PS, and further demonstrates that time‐resolved mass spectrometry is a useful tool in mechanistic studies of enzymatic reactions. Copyright © 2010 John Wiley & Sons, Ltd.