Investigation of enzyme kinetics using quench-flow techniques with MALDI-TOF mass spectrometry.
Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is combined off-line with rapid chemical quench-flow methods to investigate the pre-steady-state kinetics of a protein-tyrosine phosphatase (PTPase). PTPase kinetics are generally interrogated spectrophotometrically by the employment of an artificial, chromophoric substrate. However, that methodology places a constraint on the experiment, hampering studies of natural, biochemically relevant substrates that do not incorporate a chromophore. The mass spectrometric assay reported herein is based on the formation of a covalent phosphoenzyme intermediate during substrate turnover. This species is generated in the reaction regardless of the substrate studied and has a molecular weight 80 Da greater than that of the native enzyme. By following the appearance of this intermediate in a time-resolved manner, we can successfully measure pre-steady-state kinetics, regardless of the incorporation of a chromophore. The strengths of the mass-spectrometric assay are its uniform response to all substrates, simple and direct detection of covalent enzyme-substrate intermediates, and facile identification of enzyme heterogeneities that may affect enzymatic activity.
Department of Chemistry, Indiana University, Bloomington 47405, USA.
C T
CT
Houston
W P
WP
Taylor
T S
TS
Widlanski
J P
JP
Reilly
D002851:Chromatography, High Pressure Liquid; D004798:Enzymes / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D010449:Peptide Mapping; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D004798:Enzymes; D017027:Protein Tyrosine Phosphatases