Escherichia coli uridine diphosphate galactose 4-epimerase: circular dichroism of the protein and protein bound dihydronicotinamide adenine dinucleotide.

The circular dichroism spectra of E. coli UDP-galactose-4-epimerase in its native (epimerase-NAD+) and reduced (epimerase-NADH.UMP) forms between 190 and 400 nm are presented. The reduced form exhibits a large positive circular dichroism band at 340 nm attributed to NADH in the complex. Relative to the small negative band exhibited at this wavelength by free NADH itself, the rotational strength of enzyme-bound NADH is some 50 times larger than that of free NADH, while the oscillator strengths and other special characteristics are similar. This enhancement reflects dissymetric interactions involving the 340-nm transition and is most consistent with the dihydropyridine ring of NADH being highly immobilized in the reduced complex. In the 200- to 230-nm region both enzyme forms exhibit a negative band at 220 nm and a negative shoulder at 208 nm. The ellipticities of the reduced form are minimally 7% greater at both band positions than those of the native form. The spectra are interpreted to indicate that conversion of the native to the reduced form is accompanied by an increase in alpha-helix structure at the expense of unordered structure.