Modification of the allosteric activator site of Escherichia coli ADP-glucose synthetase by trinitrobenzenesulfonate.

Limited modification of Escherichia coli B ADP-glucose synthetase (EC 2.7.7.27) by trinitrobenzenesulfonate (TNBS) appeared to affect primarily the allosteric properties of the enzyme. There was little loss of the catalytic activity assayed in the absence of activator. However, the abilities of fructose 1,6-bisphosphate or hexanediol 1,6-bisphosphate to activate the enzyme, or of 5'-adenylate to inhibit the enzyme, were rapidly lost upon trinitrophenylation. Modification progressively decreased the affinity for activator, decreased the Vmax at saturating concentrations of activator, and decreased the cooperativity among activator binding sites. These effects could be completely prevented by the presence of allosteric effectors during reaction with TNBS, although a low amount of trinitrophenylation still occurred. Substrates partially protected the enzyme from reaction with TNBS. The lysyl epsilon-amino side chain was modified by trinitrophenylation, but the target was not primarily the same residue which could form a Schiff base with pyridoxal phosphate, another activator of the enzyme. A large peptide containing most of the trinitrophenyl residue was isolated after cleavage of the enzyme and was identified as part of the N-terminal amino acid sequence. The migration of the enzyme on polyacrylamide gel electrophoresis or on agarose column chromatography was unchanged by modification. However, the ability of fructose-1, 6-P2 to induce the oligomerization of a mutant form of the enzyme was completely prevented by trinitrophenylation. This effect could be protected against by the presence of activator or inhibitor during reaction with TNBS.