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#RobSelects preprint of the week #ChemRxiv: Determination of heavy-atom competitive kinetic isotope effects via matrix assistet laser desorption ionization time of flight mass spectrometry. #physchem https://doi.org/10.26434/chemrxiv-2023-sw8wd
ChemRxivDirect Competitive Kinetic Isotope Effect Measurement Using Quantitative Whole Molecule MALDI-TOF Mass SpectrometryKinetic isotope effect (KIE) measurements are a powerful tool to interrogate the microscopic steps in enzyme catalyzed reactions and can provide detailed information about transition state structures. However, the application of KIE measurements to study enzymatic reactions is not widely applied due to the tedious and complex analytical workflows required to measure KIEs with sufficient precession. Here, we report a method for the direct measurement of competitive KIEs using a whole molecule matrix assisted laser desorption ionization (MALDI) time of flight (TOF) mass spectrometry (MS). Using isotope labeled internal standard introduced when quenching the enzyme reaction at multiple time points enables the simultaneous measurement of both the relative heavy/light isotope ratio R and fractional conversion F relative to the internal standard for each sample as the reaction progresses. We applied this approach to measure both [1'-13C]lactose and [6'-13C]lactose KIEs for the E. coli β-galactosidase (LacZ) catalyzed hydrolysis of lactose. This MALDI-TOF MS based KIE approach can measure enzymatic KIEs with precision comparable to those obtained using competitive radioisotope labelling, and NMR based approaches.