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 site and induce a protein conformational change that increases the chemical reactivity of NAD+ at the coenzyme site. Activation of NAD+ by uridine nucleotides perturbs the N-15 and C-13 NMR chemical shifts of selectively enriched NAD+ bound to the coenzyme site. The proton-decoupled N-15 NMR signal for enzyme-bound [carboxamide-N-15]NAD+ does not change upon addition of UDP, indicating that activation is not brought about 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 shifted downfield 3.4 ppm downfield by the binding of UDP at the substrate site. These changes are consistent with the induction of a distortion into the nicotinamide ring, in which positive charge is transferred from N-1 to C-4. The kinetic and thermodynamic effects of these perturbations are significant, as indicated by the nonenzymatic chemical reactivities of a series of N-alkyl nicotinamides differing in the inductive electron withdrawing effects of the alkyl substituents. A downfield change of 3.4 ppm in the 4-C-13 chemical shifts brought about by electron withdrawal in the model compounds corresponds to a 3200-fold increase in the rate of reduction by NaBH3CN in water, a 15 000-fold increase in 86% ethanol, and a 152 mV more positive reduction potential in this series. The distortion of NAD+ by the binding of UDP is a long-range effect that is transmitted from the substrate binding site to the coenzyme through the protein conformational change. This apparently distorts the pi-electron distribution in the nicotinamide ring and reduces the activation energy for its reduction. Activation of enzyme-bound NAD+ toward reduction apparently arises from a destabilization in the nicotinamide ring structure rather than from a stabilization of the transition state through attractive interactions between the nicotinamide ring and the enzyme.