Anthranilate Synthetase of Acinetobacter calcoaceticus

Anthranilate synthetase of Acinetobacter calcoaceticus is a multimeric protein made up of two nonidentical subunits. It is not associated with anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferase, the second enzyme of the tryptophan pathway. Two classes of mutants lacking anthranilate synthetase activity are described. One is defective in the chorismate-binding subunit (E), although a normal glutamine-binding subunit (G) is produced. This class is incapable of catalyzing the anthranilate synthetase reaction. The second class is defective in the G subunit but produces an active E subunit. The second type of mutation results in a requirement for p-aminobenzoate in addition to anthranilate, suggesting a dual role for the G subunit in anthranilate and p-aminobenzoate synthesis. When extracts of the two mutant classes are mixed, normal E and G subunits can reassociate to form an active complex able to catalyze the synthesis of anthranilate. Wild type anthranilate synthetase complex is readily dissociated into E and G subunits by gel filtration through Sephadex G-100. Glycerol and glutamine stabilize the enzyme, glycerol affording complete protection while glutamine is less active. In addition to stabilizing enzyme activity, 30% glycerol enhances the association of subunits during gel filtration. Molecular weights of the individual subunits estimated from Sephadex G-100 column chromatography are 70,000 and 14,000 for the E and G subunits, respectively. The molecular weight of the anthranilate synthetase complex estimated in the presence of 30% glycerol is 86,000. Anthranilate synthetase subunits from A. calcoaceticus are capable of reassociating with those of Pseudomonas putida to form active hybrids which are approximately 50% as efficient as the native A. calcoaceticus enzyme in catalyzing anthranilate formation.