Online quench-flow electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry for elucidating kinetic and chemical enzymatic reaction mechanisms.
We have developed an automated quench-flow microreactor which interfaces directly to an electrospray ionization (ESI) mass spectrometer. We have used this device in conjunction with ESI Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) to demonstrate the potential of this approach for studying the mechanistic details of enzyme reactions. For the model system chosen to test this device, namely, the pre-steady-state hydrolysis of p-nitrophenyl acetate by the enzyme chymotrypsin, the kinetic parameters obtained are in good agreement with those in the literature. To our knowledge, this is the first reported use of online quench-flow coupled with FTICR MS. Furthermore, we have exploited the power of FTICR MS to interrogate the quenched covalently bound enzyme intermediate using top-down fragmentation. The accurate mass capabilities of FTICR MS permitted the nature of the intermediate to be assigned with high confidence. Electron capture dissociation (ECD) fragmentation allowed us to locate the intermediate to a five amino acid section of the protein--which includes the known catalytic residue, Ser(195). This experimental approach, which uniquely can provide both kinetic and chemical details of enzyme mechanisms, is a potentially powerful tool for studies of enzyme catalysis.
Clarke
David J
DJ
SIRCAMS, School of Chemistry, University of Edinburgh, West Mains Road, Edinburgh, EH9 3JJ, UK. dave.clarke@ed.ac.uk
Stokes
Adam A
AA
Langridge-Smith
Pat
P
Mackay
C Logan
CL
D000595:Amino Acid Sequence; D002918:Chymotrypsin / Q000737:chemistry / Q000378:metabolism*; D017357:Cyclotrons; D005583:Fourier Analysis; D007700:Kinetics; D008969:Molecular Sequence Data; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D002918:Chymotrypsin