Enzymes of the Tryptophan Synthetic Pathway in Brevibacterium flavun

Abstract

When tryptophan auxotrophs derived from Brevibacterium flavum which lacked tryptophan synthetase-A (TS-A), tryptophan synthetase-B (TS-B) or anthranilate-phosphoribosylpyro-phosphate phosphoribosyltransferase (PRT), were cultured in media containing limiting or excess tryptophan, the specific activities of all six tryptophan enzymes were much higher under conditions of limiting tryptophan than under conditions of excess tryptophan. This indicates that the formation of all the tryptophan enzymes was repressed by tryptophan. However, the ratio of specific activities under the two conditions was not constant among these enzymes. When the rates of synthesis of the enzymes during tryptophan starvation were compared, those of anthranilate synthetase (AS), N-5′-phosphoribosylanthranilate isornerase (PRAI), TS-A and TS-B were coordinate, while the specific activity of indole-3-glycerol phosphate synthetase (InGPS) did not increase significantly. In order to examine the molecular states of tryptophan enzymes, enzyme extracts of tryptophan auxotrophs were subjected to gel filtration on a Sephadex G-200 column. The elution profiles gave four distinct peaks; AS, PRT, PRAI and InGPS, TS-A and TS-B. Although PRAI and InGPS activities were completely coeluted in the gel filtration, they were partially dissociated on DEAE-cellulose column chromatography, giving three peaks of activity for PRAm, InGPS, and PRAI plus InGPS. TS-A and TS-B were only slightly separated in Sephadex G-200 gel filtration with glycerol. On the other hand, in the gel filtration without glycerol, neither TS-A nor TS-B activity was detected in the eluate. However, when TS-A activity was assayed in the presence of TS-B, TS-A was detected but showed a much lower molecular weight than that observed in the gel filtration with glycerol. This suggested that TS-A and TS-B were dissociated in the absence of glycerol. That is, among the six tryptophan enzymes, two dissociable enzyme complexes were observed; one in cluded TS-A and TS-B, the other PRAI and InGPS.