Measuring Kinetic Isotope Effects in Enzyme Reactions Using Time-Resolved Electrospray Mass Spectrometry
| Autor: | Peter Liuni, Ekaterina Olkhov-Mitsel, Derek J. Wilson, Arturo Orellana |
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| Rok vydání: | 2013 |
| Předmět: |
Models
Molecular Spectrometry Mass Electrospray Ionization Protein mass spectrometry Chemistry Acylation Hydrolysis Electrospray ionization Kinetics Alcohol Dehydrogenase Extractive electrospray ionization Equipment Design Mass spectrometry Sample preparation in mass spectrometry Transition state Analytical Chemistry Isotopes Computational chemistry Yeasts Kinetic isotope effect Chymotrypsin Oxidation-Reduction Enzyme Assays |
| Zdroj: | Analytical Chemistry. 85:3758-3764 |
| ISSN: | 1520-6882 0003-2700 |
| Popis: | Kinetic isotope effect (KIE) measurements are a powerful tool for studying enzyme mechanisms; they can provide insights into microscopic catalytic processes and even structural constraints for transition states. However, KIEs have not come into widespread use in enzymology, due in large part to the requirement for prohibitively cumbersome experimental procedures and daunting analytical frameworks. In this work, we introduce time-resolved electrospray ionization mass spectrometry (TRESI-MS) as a straightforward, precise, and inexpensive method for measuring KIEs. Neither radioisotopes nor large amounts of material are needed and kinetic measurements for isotopically "labeled" and "unlabeled" species are acquired simultaneously in a single "competitive" assay. The approach is demonstrated first using a relatively large isotope effect associated with yeast alcohol dehydrogenase (YADH) catalyzed oxidation of ethanol. The measured macroscopic KIE of 2.19 ± 0.05 is consistent with comparable measurements in the literature but cannot be interpreted in a way that provides insights into isotope effects in individual microscopic steps. To demonstrate the ability of TRESI-MS to directly measure intrinsic KIEs and to characterize the precision of the technique, we measure a much smaller (12)C/(13)C KIE associated specifically with presteady state acylation of chymotrypsin during hydrolysis of an ester substrate. |
| Databáze: | OpenAIRE |
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