The cooperative behavior of yeast D-glyceraldehyde-3-phosphate. Dehydrogenase as studied by the formation of the fluorescent NAD derivative.

The ultraviolet irradiation of the yeast D-glyceraldehyde-3-phosphate dehydrogenase carboxymethylated at the active site Cys residues, as with the rabbit muscle enzyme, led to the formation of a fluorescent NAD derivative with an emission maximum at 410 nm. Similar results were obtained with the enzyme selectively carboxymethylated at only 2 of its 4 active site Cys residues. The binding of NAD+ to both the carboxymethylated enzymes is non-cooperative or only weakly negatively cooperative when determined by NAD+ quenching of the intrinsic protein fluorescence. However, determinations of the amount of fluorescent NAD derivative formed under different NAD+ concentrations show that both the carboxymethylated enzymes appeared to bind NAD+ with positive cooperativity as in the case of the binding of NAD+ to the native apoenzyme. This seems to suggest that the spatial positioning of the nicotinamide moiety at the active site of the irradiated enzyme resembles more closely that of the nicotinamide ring in the native holoenzyme as compared to the carboxymethylated enzymes.