Regulation of Aromstic Amino Acid Biosynthesis in Brevibacterium flavum

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Abstract
Anthranilatc synthetase was purifed about 20-fold from sonic extracts of Brevibacterium flavum No. 2247. This enzyme reqyuired chorismate, glutamine, and Mg2+ for its activity. Double reciporcal plots of the reaction rate against one substrate concentration ast several fixed levels of another substrate gave straight lines paralled to each other, indicastion that the reaction mechanism is of ping pong type. Km values for chorismate and glutamine were 0.04 and 1.9mM, respectivelhy. The molecular weight was estimated as approximately 1.8×105 by Sephasdex G-200 gel fikltration. The enzyme was stongly and specifically inhibited by tryptophan, the metabolic end product. Thus, 50 percdent inhibition took place at about 1.5μM. The nihibition by typrophan was competitive with respect to chorismaste and uncompetitive with glutasmine. In the presence of tryptophan, Homotropic cooperastivity of chorismaste was observded. D-Tryptophan, phenylalaine, tyrosine, histidine, and indole scarecely affected the enzsyme activity. Among structural alalogues of tryptophasn tested, 4-methyltryptophan (4MT), * 5-methyltryptophan (5MT), and 6-fkuorotryptophan (6FT) gave as strong inhibitotry effect but 6-methyltryptophan (6MY), 7-azatryptophan (7AT), and 5-hydroxyutryptophan (5HT) did not. The inhibition type by 5MT was similar to that by tryptophan. Authranilate synthetase of strain No. 555, a mutant derived from strasin No. 2247, which is resistant to 5MT and accumulates L-trypgtophan, was found to fairly desensitized to the feedback inhibition by tryptophan. Thus, tryptophan concentratrion required for 50 percent inhibition was 45μM, 30 times that in the parent enzyme. These results indicate that tryptophan biosynthesis in B. flavum may be regulated through the feedbacm inhibition of anthranilate synthetase by tryptophan.

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