Primary and secondary deuterium isotope effects on equilibrium constants for enzyme-catalyzed reactions

Primary deuterium equilibrium isotope effects for the reaction of five secondary alcohols with nicotinamide adenine dinucleotide (DPN) to give reduced deuterionicotinamide adenine dinucleotide (DPND) (cyclohexanol-1-d, 1.18; 2-propanol-2-d, 1.175; threo-DL-isocitrate-2-d, 1.168; L-malate-2-d, 1.173; L-lactate-2-d, 1.19) are all approximately 1.18, while for a primary alcohol, ethanol, the value is 1.07, for an amino acid, L-glutamate-2-d, it is 1.14, and for a hemiacetal, glucose-1-d, it is 1.28. In each case deuterium becomes enriched in the alcohol, amino acid, or hemiacetal with respect to DPNH (TPNH). beta-Secondary equilibrium isotope effects for reduction of ketones by DPNH (cyclohexanone-2,2,6,6-d4, 0.82; acetone-d6, 0.78; pyruvate-d3, 0.83; alpha-ketoglutarate-3,3-d2 reduced to glutamate, 0.898; oxaloacetate-3,3-d2, 0.877; oxaloacetate-3R-d, 0.945) give an average value of 0.946/D, with deuterium becoming enriched in the alcohol or amino acid with respect to the ketone. For reduction of acetaldehyde-1-d by DPNH, the observed value of 0.953 includes the equilibrium effect on the hydration equilibrium in addition to that on the reduction, and the calculated values for reduction of the free aldehyde and the hydrate are 0.78 and 1.07. For reduction of benzaldehyde-1-d, which is not hydrated, the observed value was 0.79. The secondary equilibrium isotope effect for conversion of DPN-4-d to DPNH is 0.89, with deuterium becoming enriched in DPNH, and, for conversion of fumarate-2,3-d2 to malate, the value is 0.69, with deuterium becoming enriched in L-malate. The equilibrium isotope effect for reaction of cyclohexanol-1-d with DPN is temperature independent over the range 15-35 degrees C.