BINDING OF NAD+ TO RABBIT-MUSCLE GLYCERALDEHYDEPHOSPHATE DEHYDROGENASE AS STUDIED BY OPTICAL ROTATORY DISPERSION AND CIRCULAR DICHROISM

1. 1. The circular-dichroism spectra of both charcoal-treated and NAD+-containing glyceraldehydephosphate dehydrogenase from rabbit muscle were determined from 200 mμ to 500 mμ. 2. 2. The difference in the optical rotatory dispersion of the holoenzyme and the apoenzyme was calculated from the circular-dichroism spectra by the Kronig-Kramers transformation. 3. 3. The change in the Moffitt-Yang parameter, b0, and the change in molecular ellipticity at 350 mμ of the enzyme were followed as a function of added NAD+. The curves obtained gave a linear increase up to 2 molecules of NAD+ per molecule of enzyme, and an intersection point at 3.0 molecules NAD+ per molecule enzyme. 4. 4. By following the change in molecular ellipticity at 330 mμ on addition of 3-acetylpyridine-adenine dinucleotide, a similar result was obtained as with NAD+. 5. 5. Circular dichroism curves with a maximum at 330 mμ were found both with the enzyme-NADH complex and the enzyme-NAD+ complex treated with iodoacetate. 6. 6. Since binding of the enzyme with NAD+ has no effect on the optical rotatory dispersion or the circular dichroism below 250 mμ, it is concluded that the apo- and holoenzyme do not differ in overall protein conformation. The changes found above 250 mμ are at least partly due to extrinsic bands of the NAD+. 7. 7. The broad band between 300 and 450 mμ in the circular-dichroism spectrum of holoenzyme is probably composed of at least two components: (i) the charge-transfer complex between NAD+ and the reactive -SH group of the protein; (ii) an interaction between the adenine moiety and the enzyme. © 1969.