Measurement of kinetic isotope effects (KIEs) is a well-established and powerful tool to study enzyme reaction mechanisms and elucidating transition states. The accurate measurement of KIEs for heavy atoms surrounding the reaction center requires sensitive, precise and quantitative measurements. The widespread use of KIEs to investigate enzyme mechanisms is limited by current methods due to the lack of availability of suitable radioisotope labeled substrates or the requirement for large quantities of multiple isotope labelled substrates. Here, we report a method for the direct measurement of competitive KIEs using a whole molecule MALDI-TOF mass spectrometry. The approach uses isotope labeled internal standards introduced during reaction quenching to improve quantitative measurement of substrate concentrations and isotope ratios. This approach reduces the amount of isotope labeled material required for analysis without the need for purification prior to analysis, making it applicable to a wide variety of enzymatic reactions. We have shown the effectiveness of the method with β-galactosidase and α-glucosidase enzymes and further applied this approach to measure 1-<sup>13</sup>C KIEs for the glycosyltransferase BshA required for bacillithiol biosynthesis in Gram-positive firmicutes that plays a rrole in Fosfomycin antibiotic resistance.

New methods for enzymatic KIE measurements and TS analogue inhibitor design

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ACS Fall 2022

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