INTERACTION OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE WITH LOW-MOLECULAR-WEIGHT THIOLS

Oxidation of glyceraldehyde-3-phosphate catalyzed by glyceraldehyde-3- phosphate dehydrogenase in the absence of inorganic phosphate results in the formation of 3-phosphoglyceroyl-enzyme NADH complex which is hy drolyzed following the replacement of NADH by NAD. The hydrolysis is s trongly inhibited in the presence of low-molecular-weight thiols (cyst eine, reduced glutathione, dithiothreitol) added at concentrations com parable with the concentration of the enzyme active sites. Using [S-35 ]cysteine, the binding of the low-molecular-weight thiol to the enzyme was demonstrated, the maximal stoichiometry being 4 cysteine molecule s per enzyme tetramer. The presence of NAD and glyceraldehyde-3-phosph ate, which ensures formation of the acylated enzyme form, was found to be a prerequisite for the binding. The pH dependence of [S-35]cystein e binding is similar to the pH dependence of the effect of reduced glu tathione on the rate of 3-phosphoglyceroyl-enzyme hydrolysis. The resu lts are interpreted according to the hypothesis that covalent binding of a low-molecular-weight thiol coupled with the catalytic conversion in the enzyme active center changes the conformational state of the 3- phosphoglyceroyl-enzyme NAD complex.