PERMEABLE MEMBRANE/MASS SPECTROMETRIC MEASUREMENT OF AN ENZYMATIC KINETIC ISOTOPE EFFECT: alpha -CHYMOTRYPSIN-CATALYZED TRANSESTERIFICATION.

Article abstract of DOI:10.1021/ja00361a032

The **1**6O/**1**8O kinetic isotope effect (KIE) associated with an alpha -chymotrypsin-catalyzed transesterification was measured by using the technique of permeable membrane/mass spectroscopy. This almost in situ method is based upon the ability of the ester product, but not water and other polar compounds, to permeate through a dimethyl silicone membrane, which separates the aqueous reaction solution from the evacuated inlet of the mass spectrometer. The time course of both **1**6O and **1**8O product formation can be followed simultaneously, permitting rapid KIE measurements. With **1**8O-substituted ethanol, the KIE for the formation of both ethyl 2-furoate and ethyl 5-n-propyl-2-furoate from the respective p-nitrophenyl esters is initially normal and decreases with time to reach within approximately 2 min at 25 degree C, pH 8. 5, a constant value of 1. 009 plus or minus 0. 007 for the former and 0. 90 plus or minus 0. 02 for the latter. The large inverse steady-state KIE associated with the formation of ethyl 5-n-propyl-2-furoate decreases when the temperature is raised or when the pH is lowered.