Primary Deuterium Kinetic Isotope Effects From Product Yields: Rationale, Implementation, and Interpretation

A simple and convenient method is described to determine primary deuterium kinetic isotope effects (1 degrees DKIEs) on reactions where the hydron incorporated into the reaction product is derived from solvent water. The 1 degrees DKIE may be obtained by 1H NMR analyses as the ratio of the yields ofdH-anddD-labeled products from a reaction in 50: 50 (v/v) HOH/DOD. The procedures for these H-1 NMR analyses are reviewed. This product deuterium isotope effect (PDIE) is defined as 1/phi(EL) for fractionation of hydrons between solvent and the transition state for the reaction examined. When the solvent is not the direct hydron donor, it is necessary to correct the PDIE for the fractionation factor.EL for partitioning of the hydron between the solvent and the direct donor EL. This method was used to determine the 1 degrees DKIE on decarboxylation reactions catalyzed by wild-type orotidine 50-monophosphate decarboxylase (OMPDC) and by mutants of OMPDC, and then in the determination of the 1 degrees DKIE on the decarboxylation reaction catalyzed by 5-carboxyvanillate decarboxylase. The experimental procedures used in studies on OMPDC and the rationale for these procedures are described.