Uridine(5′)diphospho(1)α-d-galactose (UDP-gal) provides all galactosyl units in biologically synthesized carbohydrates. All healthy cells produce UDP-gal from uridine(5′)diphospho(1)α-d-glucose (UDP-glc) by the action of UDP-galactose 4-epimerase (GalE). This Account provides our recent results describing unusual mechanistic features of this enzyme.Fully active GalE is dimeric and contains one tightly bound nicotinamide adenine dinucleotide (NAD) per subunit. The NAD undergoes reversible reduction to NADH in the chemical mechanism. GalE displays unusual enzymological, chemical, and stereochemical properties. These include practically irreversible binding of NAD, nonstereospecific hydride transfer, uridine nucleotide-induced activation of NAD, Tyr149 as a base catalyst, and [GalE-NADH]-oxidation in one-electron steps by one-electron acceptors.Early studies revealed that uridine(5′) diphospho(1)α-d-4-ketopyranose (UDP-4-ketopyranose) and NADH are reaction intermediates. Weak binding of the 4-ketopyranosyl moiety and strong binding of the UDP-moiety allowed either face of the 4-ketopyranosyl moiety to accept hydride from NADH.In crystal structures of GalE, NAD bound within a Rossmann-type fold and uridine nucleotides within a substrate domain. Structures of [GalE-NADH] in complex with UDP-glc show Lys153, Tyr149, and Ser124 in contact with NAD or glucosyl-C4(OH). Lys153 forms hydrogen bonds to the ribosyl-OH groups of NAD. The phenolate of Tyr149 is associated with both the nicotinamide ring of NAD and glucosyl-C4(OH). Ser124 is hydrogen-bonded to glucosyl-C4(OH). Spectrophotometry studies show a pH-dependent charge transfer (CT) complex between Tyr149 and NAD. The CT-complex has a pKa of 6.1, which results in bleaching of the CT-band. The CT-band also bleaches upon binding of a uridine nucleotide. Kinetic experiments with wild-type GalE and Ser124Ala-GalE show the same kinetic pKa values as the corresponding CT-band pKa, which point to Tyr149 as the base catalyst for hydride transfer.We used NMR studies to verify that uridine nucleotide binding polarizes nicotinamide π-electrons. The binding of uridine(5′)-diphosphate (UDP) to GalE-[nicotinamide-1-15N]NAD shifts the 15N-signal upfield 3 ppm, whereas UDP-binding to GalE-[nicotinamide-4-13C]NAD shifts the 13C-signal downfield by 3.4 ppm. Electrochemical and 13C NMR data for a series of N-alkylnicotinamides show that the 3.4 ppm downfield 13C-perturbation in GalE corresponds to an elevation of the NAD reduction potential by 150 mV. These results account for the uridine nucleotide-dependence in the reduction of [GalE-NAD] by glucose or NaBH 3CN.Kinetics in the reduction of Tyr149Phe- and Lys153Met-GalE-NAD implicate Tyr149 and Lys153 in the nucleotide-dependent reduction of NAD. They further implicate electrostatic repulsion between N1 of NAD and the ε-aminium group of Lys153 in nucleotide-induced activation of NAD.In an O2-dependent reaction, [GalE-NADH] reduces the stable radical UDP-TEMPO with production of superoxide radical. The reaction must proceed by way of a NAD-pyridinyl radical intermediate.