Rat skeletal muscle glyceraldehyde-3-phosphate dehydrogenase: adenine nucleotide-induced inactivation.

Glyceraldehyde-3-phosphate dehydrogenase (d-glyceraldehyde-3-phosphate:nicotinamide adenine dinucleotide oxidoreductase (phosphorylating), EC 1.2.1.12), isolated from rat skeletal muscle undergoes a rapid inactivation upon incubation at 25 °C in the presence of adenine nucleotides. The reaction can be described as a reversible tetramerdimer equilibrium, only the tetrameric form of the enzyme being active in the presence of nucleotides. The standard free energy changes upon dissociation at 25 °C in 0.1 m phosphate buffer pH 7.5 in the presence of saturating concentrations of ATP, ADP, AMP, and ADP-ribose were found to be 6.69, 6.93, 8.31, and 10.5 kcal/mol, respectively. Nucleotide-dependent inactivation does not bring about any alteration of the reactivity of SH groups of the enzyme towards 5,5′-dithiobis(2-nitrobenzoic acid). This is not the case, however, when the enzyme undergoes NaCl-induced cold inactivation, which is accompanied by an increased accessibility of SH groups. ADP and ATP protect the enzyme against cold inactivation in the presence of NaCl and decrease the enhanced reactivity of SH groups. Adenine nucleotide-induced inactivation is prevented in the presence of NAD. The protective effect is noncooperative, the extent of inactivation being dependent upon the amount of active centers free of bound coenzyme. Addition of excess NAD to the inactivated enzyme results in a complete regain of activity. A comparative study made on the rate of reforming enzyme NAD complex (followed spectrophotometrically) and the regain of activity has demonstrated that the former process is markedly more rapid than the latter. The reactivation was observed to follow second-order kinetics, which suggests that the reassociation of the inactive NAD-liganded dimers is the rate-limiting step. The data are consistent with the existence of different conformational transitions responsible for the restoration of the intersubunit contact area, catalytic activity, and thermal stability of the enzyme molecule, respectively.