[Use of a fluorescent probe for the study of the active center of D-glyceraldehyde-3-phosphate dehydrogenase].
The effect of NAD on the binding of 1-anilino-8-naphthalene sulfonate (ANS) to yeast glyceraldehyde-3-phosphate dehydrogenase has been studied using difference spectrophotometric and fluorescence techniques. Coenzyme addition causes the displacement of ANS from its complex with  the dehydrogenase, as suggested by the effect of NAD on the fluorescence of the enzyme--ANS complex, as well as on the magnitude of the difference spectrum of the complex. Adenine containing NAD fragments, adenosine, 5'-AMP, and ADP were shown to compete with ANS for the common site on the enzyme using fluorimetric technique; in the case of adenosine and 5'-AMP a direct method of analytical ultracentrifugation was also employed. The results obtained by both methods suggest the dye binding at the adenine subsite of the dehydrogenase. The interaction with ANS causes no detectable conformational changes of the protein. The fluorescence of the dye-enzyme complex increases and the emission maximum shifts to shorter wavelengths on addition of nicotinamide mononucleotide. This suggest some conformational changes to occur in the microenvironment of the bound dye in response to the interaction with the ligand in the nicotinamide subsite. The participation of the nicotinamide subsite of the active center in determining the character of conformational transitions associated with coenzyme binding to glyceraldehyde-3-phosphate dehydrogenase is discussed.
D000227:Adenine Nucleotides / Q000378:metabolism; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D005470:Fluorometry; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D009243:NAD / Q000494:pharmacology; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology
D000227:Adenine Nucleotides; D000817:Anilino Naphthalenesulfonates; D009243:NAD; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases
Ispod'zovanie fluorestsiruiushchei proby dlia izucheniia adtivnogo tsentra D-glitseral'degid-3-fosfatdegidrogenazy