Mechanism of action of pyruvate kinase. Role of sulfhydryl groups in catalytic activity as determined by disulfide interchange.

Evidence is presented that four sulfhydryl residues play a critical role in the activity of pyruvate kinase. Modification of the four most reactive sulfhydryl groups by 5,5’-dithiobis(Z-nitrobenzoate) leads to a disulfide interchange reaction in which a chemically modified but catalytically active enzyme is produced. This form, which contains 5-thio-Z-nitrobenzoate in disulfide linkage to cysteine residues, is stable at low pH or in the presence of phosphoenolpyruvate and metal ions. At neutral pH and in the absence of these compounds, the thionitrobenzoate-enzyme undergoes an intramolecular disulfide interchange involving four additional but essential sulfhydryl residues to liberate thionitrobenzoate with the formation of cystine residues. The resulting form of pyruvate kinase is catalytically inactive.