Purification and Characterization of a Multienzyme Complex in the Tryptophan Pathway of Neurospora crassa

Abstract

Abstract A multienzyme complex which carries out the anthranilate synthetase, N-(5'-phosphoribosyl)anthranilate isomerase and indole-3-glycerol phosphate synthetase reactions in the tryptophan pathway of Neurospora crassa has been purified and characterized. The complex, as prepared here, is a single homogeneous protein molecule having a sedimentation coefficient of 10.3 S and a molecular weight of 240,000. Sedimentation equilibrium studies in 6 m guanidine HCl and in 0.1% sodium dodecyl sulfate reveal that the complex consists of six 40,000 molecular weight subunits. Amino acid analysis and titration with p-chloromercuribenzoate show that the complex contains 20 sulfhydryl groups and no disulfide bridges. Titration of 12 of the sulfhydryl groups or more with p-chloromercuribenzoate leads to the quantitative dissociation of the complex into 4.4 S and 7.4 S fragments. Based on the sedimentation values, the 4.4 S fragment is a dimer and the 7.4 S fragment is a tetramer of the 40,000 molecular weight subunits. The 4.4 S fragment is itself inactive but in the presence of dithiothreitol a 7 S form of anthranilate synthetase develops as the only enzymic activity. The 7.4 S fragment on the other hand, is fully active in indole-3-glycerol phosphate synthetase and N-(5'-phosphoribosyl)anthranilate isomerase. Electrophoresis in 8 m urea shows that the 4.4 S and 7.4 S fragments consist of single but distinct polypeptides, termed here the A and the I subunits, respectively. An A2I4 model for the complex is discussed.