Novel 18O kinetic isotope effect in an α-chymotrypsin catalyzed transesterification

Reactions were started by injecting the nitrophenyl ester into a stoppered vial immersed in a constant-temperature bath (40C) containing a mixture of 16O plus 18O-ethanol, α-chymotrypsin, and buffer. As the reaction proceeded, samples were withdrawn and injected into a gas chromatograph connected to a quadrupole mass spectrometer controlled by a data acquisition and processing system. The intensities from individual ions were displayed as a function of time to yield chromatographic peaks. Relative 18O enrichments were computed from the ratios of the peak areas, and the apparent KIE was then calculated from the ratio of enrichments in alcohol and ester. The values of the plateau KIE were less than or equal to 0.90 for all experiments. The apparent first-order rate constant can also be calculated from the magnitude of the initial burst and the slope of the zero-order absorbence increase. This apparent rate constant varies linearly with the ethanol concentration permitting calculation of the second-order rate associated with the formation of the ethyl ester product. 2 figures, 1 table