PMID- 10939405
OWN - NLM
STAT- MEDLINE
DCOM- 20001017
LR  - 20190717
IS  - 0003-2700 (Print)
IS  - 0003-2700 (Linking)
VI  - 72
IP  - 14
DP  - 2000 Jul 15
TI  - Investigation of enzyme kinetics using quench-flow techniques with MALDI-TOF mass 
      spectrometry.
PG  - 3311-9
AB  - 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.
FAU - Houston, C T
AU  - Houston CT
AD  - Department of Chemistry, Indiana University, Bloomington 47405, USA.
FAU - Taylor, W P
AU  - Taylor WP
FAU - Widlanski, T S
AU  - Widlanski TS
FAU - Reilly, J P
AU  - Reilly JP
LA  - eng
PT  - Journal Article
PT  - Research Support, U.S. Gov't, Non-P.H.S.
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - Anal Chem
JT  - Analytical chemistry
JID - 0370536
RN  - 0 (Enzymes)
RN  - EC 3.1.3.48 (Protein Tyrosine Phosphatases)
SB  - IM
MH  - Chromatography, High Pressure Liquid
MH  - Enzymes/chemistry/*metabolism
MH  - Kinetics
MH  - Peptide Mapping
MH  - Protein Tyrosine Phosphatases/metabolism
MH  - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
EDAT- 2000/08/12 11:00
MHDA- 2000/10/21 11:01
CRDT- 2000/08/12 11:00
PHST- 2000/08/12 11:00 [pubmed]
PHST- 2000/10/21 11:01 [medline]
PHST- 2000/08/12 11:00 [entrez]
AID - 10.1021/ac991499m [doi]
PST - ppublish
SO  - Anal Chem. 2000 Jul 15;72(14):3311-9. doi: 10.1021/ac991499m.