Observation of a chemically labile, noncovalent enzyme intermediate in the reaction of metal-dependent Aquifex pyrophilus KDO8PS by time-resolved mass spectrometry

Rapid Commun Mass Spectrom. 2010 Jul 15;24(13):1919-24. doi: 10.1002/rcm.4594.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Aldehyde-Lyases / chemistry*
  • Aldehyde-Lyases / metabolism
  • Bacteria / enzymology*
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / metabolism
  • Cadmium / chemistry*
  • Cadmium / metabolism
  • Kinetics
  • Pentosephosphates / chemistry
  • Pentosephosphates / metabolism
  • Phosphoenolpyruvate / chemistry
  • Phosphoenolpyruvate / metabolism
  • Spectrometry, Mass, Electrospray Ionization / methods*
  • Time Factors

Substances

  • Bacterial Proteins
  • Pentosephosphates
  • Cadmium
  • arabinose 5-phosphate
  • Phosphoenolpyruvate
  • 2-dehydro-3-deoxyphosphooctonate aldolase
  • Aldehyde-Lyases