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.