Kinetic studies of the reactivity of the sulfhydryl groups of glyceraldehyde-3-phosphate dehydrogenase.
The reaction of sulfhydryl groups of glyceraldehyde-3-phosphate dehydrogenase from rabbit and pig muscles with a large molar excess of 5,5'-dithiobis(2-nitrobenzoate) (Nbs2) shows three-phasic pseudo-first-order kinetics. Since the fastest reaction between active cysteine-149 and Nbs2 is apparently biphasic, half-of-the-sites reactivity towards Nbs2 is suggested. Further sulfhydryl groups become reactive as an effect of conformational changes in the protein molecule after formation of a mixed disulfide on cysteine-149. In the presence of 40 mM borate the reaction is biphasic only, and two sulfhydryl groups per subunit react very quickly. The bound NAD+ is only partially released even after a long reaction with Nbs2. It was demonstrated that the two NAD+ binding sites with the highest dissociation constants have no significant effect on the reaction between cysteine-149 and Nbs2.
D000818:Animals; D001665:Binding Sites; D001881:Borates / Q000494:pharmacology; D004228:Dithionitrobenzoic Acid; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D009132:Muscles / Q000201:enzymology; D009994:Osmolar Concentration; D011485:Protein Binding; D011487:Protein Conformation; D013438:Sulfhydryl Compounds; D013552:Swine
D001881:Borates; D046911:Macromolecular Substances; D013438:Sulfhydryl Compounds; D004228:Dithionitrobenzoic Acid; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases