Kinetic Studies of the Reactivity of the Sulfhydryl Groups of Glyceraldehyde-3-Phosphate Dehydrogenase

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Abstract
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 threephasic 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.