Kinetic monitoring of enzymatic reactions in real time by quantitative high-performance liquid chromatography-mass spectrometry.
The study of enzyme kinetics under steady-state conditions represents a common and very useful method for investigating the mechanisms of enzymatic reactions. We report the use of mass spectrometry (MS) coupled with HPLC for the kinetic analysis of enzymatic reactions in real time. The hydrolysis of dinucleotides with bovine pancreatic ribonuclease A (RNase A) and the substrate-specific hydrolysis of lactose with beta-galactosidase can be monitored using ion-spray (pneumatically assisted electrospray) mass spectrometry as a sensitive and specific detector for the native substrates. The resulting data can be used to calculate both KM and Vmax for each system. Kinetic parameters obtained for RNase A and beta-galactosidase paralleled those obtained by conventional techniques. These findings suggest the possibility of developing alternative techniques, based on mass spectrometric detection, for performing kinetic analyses of enzymatic processes where no simple spectrophotometric assay is feasible. In addition to enabling the determination of kinetic parameters for authentic substrates, and not chromogenic analogs, such assays would also be useful in situations where very high sensitivity and specificity are desired.
Cornell University, Ithaca, New York 14850, USA.
F Y
FY
Hsieh
X
X
Tong
T
T
Wachs
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B
Ganem
J
J
Henion
D000818:Animals; D002240:Carbohydrate Sequence; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000706:statistics & numerical data; D004798:Enzymes / Q000378:metabolism*; D005069:Evaluation Studies as Topic; D066298:In Vitro Techniques; D007700:Kinetics; D007785:Lactose / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods* / Q000706:statistics & numerical data; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D012680:Sensitivity and Specificity; D001616:beta-Galactosidase / Q000378:metabolism
D004798:Enzymes; D009841:Oligonucleotides; D012259:Ribonuclease, Pancreatic; D001616:beta-Galactosidase; D007785:Lactose