title: The specificity of induced conformational changes. The case of yeast glyceraldehyde-3-phosphate dehydrogenase.
abstract: The specificity of induced conformational changes and of the probes used to detect them has been investigated in yeast glyceraldehyde-3-phosphate dehydrogenase. Cyanylation of the active-site SH groups in two of the four identical subunits of glyceraldehyde-3-phosphate dehydrogenase has no effect on reactivity of the unmodified SH groups toward the cyanylating reagent (2-nitro-5-thiocyanogenzoic acid, NTCB) but results in total loss of catalytic activity. Cyanylation of the dicarboxamidomethylated enzyme was four orders of magnitude slower than with the unmodified enzyme in contrast to cyanylation of the dicyanylated enzyme. Cyanylation by NTCB as well as alkylation by iodoacetate and acylation with beta-(2-furyl)acryloyl phosphate are enhanced in the presence of NAD+ while alkylation by iodoacetamide is inhibited by NAD+. In the absence of NAD+, hydrolysis of the acylated enzyme is faster than phosphorolysis while the reverse is true in the presence of NAD+. NAD+ accelerates hydrolysis of the 3-phosphoglyceroylated enzyme about 60-fold but decreases the rate of hydrolysis of the furylacryloylated enzyme by a factor of 17. Other examples of the specificity of the induced conformational changes and the probes are described. The conformational changes induced by NAD+ make the protein specifically reactive toward its physiological substrates and less reactive toward extraneous competing compounds.
mesh_terms: D001565:Benzoates; D001665:Binding Sites; D003545:Cysteine / Q000032:analysis; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D007460:Iodoacetamide; D007700:Kinetics; D009243:NAD; D009570:Nitriles; D011485:Protein Binding; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013861:Thiocyanates
chemical_list: D001565:Benzoates; D009570:Nitriles; D013861:Thiocyanates; D009243:NAD; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D003545:Cysteine; D007460:Iodoacetamide