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The ultraviolet irradiation of rite yeast D-glyceraldchyde-3-phosphate dehydrogenase carbox-ymethylated at the active site Cys residues, as with the rabbit muscle enzyme, led to the for-mation of a fluorcscent NAD derivative with an emission maximum at 410 mn. Similar resultswere obtained with the enzyme selectively carboxymethylated at only 2 of its 4 active site Cysresidues. The binding of NAD~+ to both the carboxymethylated enzymes is non-cooperative oronly weakly negatively cooperative when determined by NAD~+ quenching of thc intrinsic proteinfluorescence, However, determinations of the amount of fluorescent NAD derivative formedunder different NAD~+ concentrations show that both the earboxymethylated enzymes appearedto bind NAD~+ with positive cooperativity as in the ease of the binding of NAD~+ to thenative apoenzyme. This seems to suggest that the spatial positioning of the nicotinamidemoity at the active site of the irradiated enzyme resembles more closely that of the nico-tinamide ring in the
The ultraviolet irradiation of rite yeast D-glyceraldchyde-3-phosphate dehydrogenase carbox-ymethylated at the active site Cys residues, as with the rabbit muscle enzyme, led to the for mation of a fluorcscent NAD derivative with an emission maximum at 410 mn. The results of the enzyme with carboxymethylated at only 2 of its 4 active site Cysresidues. The binding of NAD ~ + to both the carboxymethylated enzymes is non-cooperative or ofly weakly negatively cooperative when determined by NAD ~ + quenching of thc intrinsic protein fluorescences, However, , determinations of the amount of fluorescent NAD derivative formed by different NAD ~ + concentrations show that both the earboxymethylated enzymes appeared bind NAD ~ + with positive cooperativity as in the ease of the binding of NAD ~ + to thenative apoenzyme. This seems to suggest that the spatial positioning of the nicotinamidemoity at the active site of the irradiated enzyme resembles more closely that of the nico-tin amide ring in the