92333-73-8Relevant academic research and scientific papers
Measuring kinetic isotope effects in enzyme reactions using time-resolved electrospray mass spectrometry
Liuni, Peter,Olkhov-Mitsel, Ekaterina,Orellana, Arturo,Wilson, Derek J.
, p. 3758 - 3764 (2013)
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 12C/ 13C KIE associated specifically with presteady state acylation of chymotrypsin during hydrolysis of an ester substrate.
