Binding of NAD + to rabbit-muscle glyceraldehydephosphate dehydrogenase. The use of [formula omitted] chloride as a spectral and conformational probe

Abstract

Addition of N- methylnicotinamidium chloride to rabbit-muscle glyceraldehydephosphate dehydrogenase ( d-glyceraldehyde-3-phosphate:NAD + oxidoreductase (phosphorylating), EC 1.2.1.12) results in the appearance of a charge-transfer absorption spectrum, with an apparent maximum around 327 nm, that is insensitive to the sulfhydryl reagents iodoacetate and p- chloromercurisulfonate . Resolution of this band and of the Racker band, resulting from the binding of NAD + to the enzyme, gives two overlapping Gaussian absorption bands with maxima at the same wavelength (324 and 369 nm) but with different relative intensities for the two complexes. From these data it is concluded that tryptophan is involved as the electron-donating group in the charge-transfer absorption band of the enzyme-NAD + complex. The different relative intensities of the two overlapping Gaussian absorption bands are attributed to dissimilarities in the relative orientation of the nicotinamidium ion of NAD + and N- methylnicotinamidium chloride, respectively, with respect to the tryptophan molecule involved. By using the charge-transfer absorption band of the enzyme-N- methylnicotinamidium complex as a conformational probe, it is shown that the effect on the protein conformation, caused by the binding of the first two NAD + molecules to the enzyme, is different from that brought about by the binding of the third and fourth NAD + molecule.