Measuring Kinetic Isotope Effects in Enzyme Reactions Using Time-Resolved Electrospray Mass Spectrometry
Measuring Kinetic Isotope Effects in Enzyme Reactions Using Time-Resolved Electrospray Mass Spectrometry
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DOI：
10.1021/ac400191t
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发表时间：
2013-04-02
影响因子：
7.4
通讯作者：
Wilson, Derek J.
中科院分区：
文献类型：
--
作者：
Liuni, Peter;Olkhov-Mitsel, Ekaterina;Wilson, Derek J.
Kinetic isotope effect (ME) 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 ME 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 C-12/C-13 KIE associated specifically with presteady state acylation of chymotrypsin during hydrolysis of an ester substrate.
