Observation of a chemically labile, noncovalent enzyme intermediate in the reaction of metal-dependent Aquifex pyrophilus KDO8PS by time-resolved mass spectrometry.
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.
Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06510, USA.
Anne
A
Roberts
Cristina
C
Furdui
Karen S
KS
Anderson
D000446:Aldehyde-Lyases / Q000737:chemistry* / Q000378:metabolism; D001419:Bacteria / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002104:Cadmium / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D010428:Pentosephosphates / Q000737:chemistry / Q000378:metabolism; D010728:Phosphoenolpyruvate / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013997:Time Factors
D001426:Bacterial Proteins; D010428:Pentosephosphates; D002104:Cadmium; C028055:arabinose 5-phosphate; D010728:Phosphoenolpyruvate; C020011:2-dehydro-3-deoxyphosphooctonate aldolase; D000446:Aldehyde-Lyases