The importance of binding energy in catalysis of hydride transfer by UDP-galactose 4-epimerase: A carbon-13 and nitrogen-15 NMR and kinetic study

Abstract

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.

On the Enzymatic Activation of NADH

“…Theoretical calculations on the nicotinamide indicated that deformation of the pyridine ring into a boat conformation enhances hydride transfer (2). It has been observed by UV-visible spectroscopy and NMR that the puckering of the pyridine ring changes upon enzyme binding (3). Until now it was unclear how an enzyme could steer the puckering of the ring to facilitate hydride transfer.…”

Changing the Donor Cofactor of Bovine α1,3-Galactosyltransferase by Fusion with UDP-galactose 4-Epimerase

“…It was reported that each subunit of the dimeric epimerase contained one irreversibly, noncovalently bound NAD ϩ (33), and no additional NAD ϩ was required for the epimerase-catalyzed reaction (32). This is also the case for the fusion enzymes.…”

“…UDP-Gal 4-epimerase is one of the key enzymes of the Leloir pathway for galactose metabolism (32). The epimerase from E. coli is a homodimer with an overall molecular mass of 79 kDa (33).…”

Transient Intermediates in Enzymology, 1964–2008

“…Biological phosphates, being achiral or prochiral at tetrahedral phosphorus, had to be synthesized with P-substituents such as S, 18 O, and/or 17 O stereospecifically placed to study stereochemistry (22). The ping-pong mechanism of GalT implied overall retention of configuration at P␣ of UDP-glucose through stereochemical inversion in two steps.…”

“…Our laboratory developed 31 In stereochemical work on phosphotransferases, Pchiral adenosine 5Ј-O-(3- [3-thioate groups in the products were determined by chemical, mass spectrometric, or 31 P NMR methods. R. Douglas Sammons, Thomas Meade, and Radha Iyengar devised methods for displacing sulfur from P-chiral phosphorothioate esters with 18 O or 17 O from water (25, 26). Our group applied these methods to elucidate the stereochemical course of phosphotransfer ( Table 1).…”