NAD+ is very tightly but noncovalently bound in the active site, NAD+ is reduced to NADH in the course of catalysis. NADH associated with the purified enzyme is a component of the inactive, abortive complexes, enzyme-NADH-uridine nucleotide, that contain tightly bound uridine nucleotides in place of the epimerization intermediate UDP-4-keto-alpha-D-hexoglucopyranose. These complexes are produced in vivo in the course of bacterial growth
coenzyme is tightly bound at the active site. NAD+ functions as the coenzyme for the interconversion of UDP-galactose and UDP-glucose by reversibly mediating their dehydrogenation to the common intermediate UDP-4-ketohexopyranoside. NAD+ activation induced by uridine nucleotides is brought about by a conformational change of epimerase that repositions Tyr149 at an increased distance from nicotinamide N1 of NAD+ while maintaining the electrostatic repulsion between Lys153 and nicotinamide N1 of NAD+
1.78 mol of NAD+ per dimer. Each subunit is independently capable of being associated with one molecule of NAD+, suggestive of two NAD+ binding sites of epimerase per dimer
the fully active GalE is dimeric and contains one tightly bound NAD+ per subunit, NAD+ undergoes reversible reduction to NADH in the chemical mechanism, practically irreversible binding of NAD+ within a Rossmann-type fold, 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. pH-Dependent charge transfer complex between Tyr149 and NAD+. Binding structure, overview
enzyme-bound, exogenous NAD+ does not appear to be a strong activator of enzyme activity, there is no significant difference in enzyme activity regardless of the presence and absence of NAD+
the NAD+ cofactor can be removed from human GalE without denaturation. Fewer protein-NAD+ contacts are observed in the crystal structure, which explains the reversible character of cofactor binding
two paired Rossmann folds tightly bind one NAD+ cofactor per subunit. In Escherichia coli GalE, the NAD+ interacts more extensively with the protein than is observed with other SDR enzymes. pH-Dependent charge transfer complex between Tyr149 and NAD+