The importance of binding energy in catalysis of hydride transfer by UDP-galactose 4-epimerase: a 13C and 15N 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 15N and 13C NMR chemical shifts of selectively enriched NAD+ bound to the coenzyme site. The proton-decoupled 15N NMR signal for enzyme-bound [carboxamide-15N]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 15N NMR signal of enzyme-bound [nicotinamide-1-15N]NAD+ is shifted upfield 3.0 ppm and the 13C NMR signal for [nicotinamide-4-13C]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 4-13C 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.
Burke
J R
JR
Institute for Enzyme Research, Graduate School, University of Wisconsin-Madison 53705.
Frey
P A
PA
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009243:NAD / Q000737:chemistry; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D014534:UDPglucose 4-Epimerase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D014530:Uridine Diphosphate / Q000737:chemistry
D001426:Bacterial Proteins; D009243:NAD; D014530:Uridine Diphosphate; D014534:UDPglucose 4-Epimerase