THE IMPORTANCE OF BINDING-ENERGY IN CATALYSIS OF HYDRIDE TRANSFER BY UDP-GALACTOSE 4-EPIMERASE - A C-13 AND N-15 NMR AND KINETIC-STUDY

UDP-galactose 4-epimerase contains NAD+ irreversibly but noncovalently bound to the active site. Uridine nucleotides bind to the substrate s ite and induce a protein conformational change that increases the chem ical reactivity of NAD+ at the coenzyme site. Activation of NAD+ by ur idine nucleotides perturbs the N-15 and C-13 NMR chemical shifts of se lectively enriched NAD+ bound to the coenzyme site. The proton-decoupl ed N-15 NMR signal for enzyme-bound [carboxamide-N-15]NAD+ does not ch ange upon addition of UDP, indicating that activation is not brought a bout by a change in the binding of the carboxamide group. The N-15 NMR signal of enzyme-bound [nicotinamide-1-N-15]NAD+ is shifted upfield 3 .0 ppm and the C-13 NMR signal for [nicotinamide-4-C-13]NAD+ is shifte d downfield 3.4 ppm downfield by the binding of UDP at the substrate s ite. These changes are consistent with the induction of a distortion i nto the nicotinamide ring, in which positive charge is transferred fro m N-1 to C-4. The kinetic and thermodynamic effects of these perturbat ions are significant, as indicated by the nonenzymatic chemical reacti vities of a series of N-alkyl nicotinamides differing in the inductive electron withdrawing effects of the alkyl substituents. A downfield c hange of 3.4 ppm in the 4-C-13 chemical shifts brought about by electr on withdrawal in the model compounds corresponds to a 3200-fold increa se in the rate of reduction by NaBH3CN in water, a 15 000-fold increas e in 86% ethanol, and a 152 mV more positive reduction potential in th is series. The distortion of NAD+ by the binding of UDP is a long-rang e effect that is transmitted from the substrate binding site to the co enzyme through the protein conformational change. This apparently dist orts the pi-electron distribution in the nicotinamide ring and reduces the activation energy for its reduction. Activation of enzyme-bound N AD+ toward reduction apparently arises from a destabilization in the n icotinamide ring structure rather than from a stabilization of the tra nsition state through attractive interactions between the nicotinamide ring and the enzyme.