The Anthranilate Synthetase-Anthranilate 5-Phosphoribosylpyrophosphate Phosphoribosyltransferase Aggregate: PURIFICATION OF THE AGGREGATE AND REGULATORY PROPERTIES OF ANTHRANILATE SYNTHETASE

Abstract

Abstract Relatively stable, highly purified preparations of the anthranilate synthetase-anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase aggregate have been obtained from Salmonella tryphimurium. A molecular weight of approximately 285,000 was estimated for the enzyme aggregate by sucrose gradient centrifugation. Anthranilate synthetase activity was completely inhibited by 10 µm tryptophan under appropriate conditions. Inhibition by tryptophan was competitive with chorismate and noncompetitive with glutamine and showed positive cooperativity. Anthranilate synthetase activity was almost completely insensitive to inhibition by tryptophan in the presence of 5.0 mm Mg2+, suggesting distinct substrate and inhibitor sites. Mg2+ was required for enzymatic activity. Inhibition of enzyme activity was obtained by low concentrations of anthranilate, indicating possible regulatory significance. This inhibition was partially overcome by excess Mg2+, suggesting that inhibition was due, at least in part, to interaction of anthranilate with the tryptophan regulatory site. Positive cooperativity obtained for anthranilate was similar to that obtained for tryptophan. In the presence of anthranilate or tryptophan positive cooperativity for chorismate was observed. For the NH3-dependent anthranilate synthetase activity of the anthranilate synthetase-anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase aggregate, negative cooperativity for NH3 was indicated by the shapes of plots of initial velocity against (NH4)2SO4 concentration and Lineweaver-Burk plots and the values for related kinetic constants. Initial velocity experiments were consistent with a sequential mechanism.