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.