Evidence for Compartmentation of Tryptophan in Cultured Plant Tissues: Free Tryptophan Levels and Inhibition of Anthranilate Synthetase

1. Anthranilate synthetase activity in crude extracts from tissue cultures of Daucus carota L. (carrot), Nicotiana tabacum L. (tobacco; cv. Wisconsin 38 and xanthi), Glycine max Merr. (soybean) and Oryza sativa L. (rice) was completely inhibited by l -tryptophan (5 to 50 μM). Mutant carrot and tobacco lines, capable of growth in the presence of 5-methyltryptophan, required 500 to more than 1000 μM tryptophan for complete inhibition of enzyme activity, respectively. 2. Except for the mutant tobacco line, the concentrations of free tryptophan in all tissue cultures tested were greater than the levels necessary to completely inhibit the respective anthranilate synthetase activities in vitro. These findings would indicate that much of the free tryptophan is compartmentalized away from the regulatory enzyme, anthranilate synthetase. This could implicate compartmentalization of the inhibitor as a biosynthetic control mechanism. 3. During the growth of normal and mutant carrot tissues the anthranilate synthetase enzyme must be at least 7.8 and 10.8% active, respectively, in order to accumulate the amount of tryptophan found in the tissues. 4. Of the substrates and cofactors required for anthranilate synthetase activity in vitro, Mg²⁺ and glutamine were present at near optimal levels in the carrot and tobacco tissues, but chorismate was found to be significantly below the optimal concentrations.     

Measurement of the Five Enzymes which Convert Chorismate to Tryptophan in Wheat Plants (Triticum aestivum cv. Kalyansona)

Conditions are described for measuring anthranilate synthetase, anthranilate-PRPP-phosphoribosyl transferase, N-5′-phosphoribosyl anthranilate isomerase, indole-3-glycerol phosphate synthetase and tryptophan synthetase in crude extracts from Triticum aestivum (wheat) plants. Only the last enzyme has been measured before in extracts from green plants. The extractable quantities of each enzyme in all plant parts at all stages of growth were sufficient to synthesize the amount of tryptophan present within the same tissue in 48 h. Anthranilate synthetase activity was the lowest of the five enzyme activities and was the only one inhibited by tryptophan in vitro, indicating that this enzyme may be the control point in tryptophan biosynthesis in wheat plants.     

Enzymes of the Tryptophan Pathway in Acinetobacter calco-aceticus

All enzymes of the tryptophan synthetic pathway were detectable in extracts from wild-type Acinetobacter calco-aceticus. The levels of these enzymes were determined in extracts from a number of auxotrophs grown under limiting tryptophan. In each case only anthranilate synthetase was found to be present in increased amounts, whereas the specific activities of the remaining enzymes remained unchanged and unaffected by the tryptophan concentration. Derepression of anthranilate synthetase was found to occur as the concentration of tryptophan became limiting. Anthranilate synthetase and phosphoribosyl transferase activities are both feedback-inhibited by tryptophan. Molecular weight determination carried out by gel filtration and zonal centrifugation in sucrose revealed that all the enzymes are less than 100,000, and no molecular aggregates of these enzymes were detected. The data indicate that tryptophan synthesis in Acinetobacter is regulated both by feedback inhibition of the first two enzymes of the pathway and by repression control of anthranilate synthetase.     

Separation of Two Forms of Anthranilate Synthetase from 5-Methyltryptophar Susceptible and -Resistant Cultured Solarium tuberosumCells

Potato cell suspension cultures (Solanum tuberosumL. cv. Merrimack) have been selected which are resistant to growth inhibition by D,L-5-methyltryptophan. Anthranilate synthetase activity in crude extracts from resistant cells was less sensitive to feedback inhibition by L-tryptophan and D,L-5-methyltryptophan than the activity from the sensitive line. This altered feedback control apparently accounts for the cell's resistance to growth inhibition since there is a 48-fold increase in free tryptophan in one of the resistant cell lines. Preparative polyacrylamide gel electro-phoresis separated feedback-sensitive and -resistant forms of anthranilate synthetase in extracts from both 5-methyltryptophan-susceptible and -resistant cells, with a predominance of the corresponding form in the respective cell type. The anthranilate synthetase activity from the 5-methyltryptophan-resistant line was inactivated more slowly by incubation of crude extracts at 50°C than the activity from the sensitive line. These results suggest the presence of two isoenzymes of anthranilate synthetase in cultured potato cells.     

Tryptophan Synthetic Pathway and Its Regulation in Chromobacterium violaceum

Extracts of Chromobacterium violaceum catalyzed all of the reactions involved in synthesizing tryptophan from chorismic acid. Tryptophan auxotrophs which had lost any of these activities did not produce the characteristic purple pigment, violacein, when grown on a medium in which tryptophan was limiting. Gel filtration of extracts allowed us to estimate molecular weights for the tryptophan enzymes. All of the enzymes appeared to have molecular weights below 100,000. No enzymes were observed to occur in aggregates. The specific activities of the enzymes of the tryptophan pathway did not change when mutants were grown under conditions of limiting or excess tryptophan. The first enzyme in the pathway, anthranilate synthetase, was subject to feedback control by the end product, tryptophan. Tryptophan acted as a noncompetitive inhibitor with respect to glutamine, one of the substrates for anthranilate synthetase, and as a competitive inhibitor of the reaction when chorismate, the other substrate, was varied. The nonlinearity observed in the Lineweaver-Burk plot in the latter case suggests that there may be more than one chorismate-binding site on anthranilate synthetase.     

Tryptophan Analog Resistance Mutations in Chlamydomonas Reinhardtii

Forty single gene mutations in Chlamydomonas reinhardtii were isolated based on resistance to the compound 5'-methyl anthranilic acid (5-MAA). In other organisms, 5-MAA is converted to 5'-methyltryptophan (5-MT) and 5-MT is a potent inhibitor of anthranilate synthase, which catalyzes the first committed step in tryptophan biosynthesis. The mutant strains fall into two phenotypic classes based on the rate of cell division in the absence of 5-MAA. Strains with class I mutations divide more slowly than wild-type cells. These 17 mutations map to seven loci, which are designated MAA1 to MAA7. Strains with class II mutations have generation times indistinguishable from wild-type cells, and 7 of these 23 mutations map to loci defined by class I mutations. The remainder of the class II mutations map to 9 other loci, which are designated MAA8-MAA16. The maa5-1 mutant strain excretes high levels of anthranilate and phenylalanine into the medium. In this strain, four enzymatic activities in the tryptophan biosynthetic pathway are increased at least twofold. These include the combined activities of anthranilate phosphoribosyl transferase, phosphoribosyl anthranilate isomerase, indoleglycerol phosphate synthetase and anthranilate synthase. The slow growth phenotypes of strains with class I mutations are not rescued by the addition of tryptophan, but the slow growth phenotype of the maa6-1 mutant strain is partially rescued by the addition of indole. The maa6-1 mutant strain excretes a fluorescent compound into the medium, and cell extracts have no combined anthranilate phosphoribosyl transferase, phosphoribosyl anthranilate isomerase and indoleglycerol phosphate synthetase activity. The MAA6 locus is likely to encode a tryptophan biosynthetic enzyme. None of the other class I mutations affected these enzyme activities. Based on the phenotypes of double mutant strains, epistatic relationships among the class I mutations have been determined.     

Enzymatic Basis for Overproduction of Tryptophan and Its Metabolites in Hansenula polymorpha Mutants

3-Deoxy-d-arabinoheptulosonate 7-phosphate (DAHP) synthetase and anthranilate synthetase are key regulatory enzymes in the aromatic amino acid biosynthetic pathway. The DAHP synthetase activity of Hansenula polymorpha was subject to additive feedback inhibition by phenylalanine and tyrosine but not by tryptophan. The synthesis of DAHP synthetase in this yeast was not repressed by exogenous aromatic amino acids, singly or in combinations. The activity of anthranilate synthetase was sensitive to feedback inhibition by tryptophan, but exogenous tryptophan did not repress the synthesis of this enzyme. Nevertheless, internal repression of anthranilate synthetase probably exists, since the content of this enzyme in H. polymorpha strain 3-136 was double that in the wild-type and less sensitive 5-fluorotryptophan-resistant strains. The biochemical mechanism for the overproduction of indoles by the 5-fluorotryptophan-resistant mutants was due primarily to a partial desensitization of the anthranilate synthetase of these strains to feedback inhibition by tryptophan. These results support the concept that inhibition of enzyme activities rather than enzyme repression is more important in the regulation of aromatic amino acid biosynthesis in H. polymorpha.     

Tryptophan synthetase in Euglena gracilis strain G

The five enzyme activities in the synthesis of l-tryptophan have been obtained in extracts of Euglena gracilis. One of these, tryptophan synthetase, has been studied in detail. The general catalytic properties of tryptophan synthetase, including the range of reactions catalyzed and its substrate and cofactor affinities, are similar to those reported for other organisms. The Euglena enzyme has two properties never previously observed for tryptophan synthetase. First, the rate of catalysis of the conversion of indole-glycerol phosphate to l-tryptophan remained at its maximal value and was unaffected by the ionic environment up to 0.3 m KCl. In contrast, the conversion of indole to tryptophan showed a sharp maximum at 0.08 m KCl. Second, the enzyme is a component of a complex that includes every enzyme in the pathway committed to tryptophan biosynthesis with the exception of anthranilate synthetase, the regulatory enzyme.     

Influences of Various Amino Acids on Tryptophan-Mediated Control of the Tryptophan Biosynthetic Enzymes in Escherichia coli

Lysates of Escherichia coli Ymel obtained from cultures grown in the absence of tryptophan in minimal medium supplemented with 0.1% casein hydrolysate show an approximate fivefold increase in steady-state specific activity of both anthranilate synthetase and tryptophan synthetase A protein relative to cultures grown in nonsupplemented medium. In the presence of repressing levels of exogenous tryptophan, growth of cultures in casein hydrolysate-supplemented medium results in a noncoordinate enhancement of repression of 10-fold for anthranilate synthetase and twofold for tryptophan synthetase A protein. Similar, but less pronounced, effects are shown for strain W3110. Strains possessing tryptophan regulator gene mutations do not exhibit this first effect, but do yield an approximate twofold decrease in specific activity of both enzymes when grown in medium supplemented with tryptophan and casein hydrolysate. A stimulation of derepression of both enzymes in strain Ymel equivalent to that induced by casein hydrolysate can be reproduced by growth in minimal medium supplemented with threonine, phenylalanine, tyrosine, serine, glutamic acid, and glutamine. Doubling time in this medium is not significantly different from that in minimal medium. An enhancement of repression which partially mimics that observed on growth in medium supplemented with tryptophan plus casein hydrolysate is obtained when Ymel is grown on medium supplemented with tryptophan plus methionine. Threonine or phenylalanine plus tyrosine as separate medium supplements are independently capable of producing a 1.4-fold or 3.4-fold stimulation, respectively, but in combination only the phenylalanine plus tyrosine effect is manifested unless serine and glutamic acid or glutamine are included. Our data show that expression of the tryptophan biosynthetic enzymes can be significantly influenced in vivo as a result of growth in medium supplemented with a variety of amino acids.     

Control of Aromatic Amino Acid Biosynthesis in Cultured Plant Tissues: Effect of Intermediates and Aromatic Amino Acids on Free Levels

Shikimate, anthranilate, indole, l -tryptophan, phenylpyruvate, l -p henylalanine, p-hydroxyphenylpyruvate or l -tyrosine were added to suspension-cultured Nicotiana tabacum (tabacco) and Daucus carota (carrot) tissues and incubated for 24 hours. Uptake of each compound was substantial as measured by its decrease in the medium. The levels of free tryptophan, phenylalanine and tyrosine were determined in the tissues after the 24 hours incubation. Shikimate did not change the aromatic animo acid levels in carrot tissue, but did increase all three in tobacco (3-fold or more), indicating a less stringent feedback control in tobacco. Anthranilate and indole increased the tissue tryptophan levels in both species by at least 17-fold, showing that the flow from anthranilate and indole to tryptophan was apparently unhindered by enzymatic control mechanisms. When tryptophan levels were elevated in both carrot and tobaccotissues by anthranilate, indole or tryptophan addition, there was also an increase in free phyenylalanine and tyrosine. This might be due to the reversal of phenylalanine and tyrosine feedback inhibition of chorismate mutase by the high tryptophan in the tissue. Chorismate mutase activity in tobacco crude extracts could be inhibited by 66–90% by 1 mM phenylalanine and /or tyrosine. Tryptophan at 1 mM stimulated the enzyme activity by 1/3 and completely reversed the phenylalanine and/or tyrosine inhibition of enzyme activity. Chorsimate mutase activity amino acids under a variety of conditions. Phenylpyruvate increased the phenylalanine levels and p-hydroxyphenylpyruvate increased the tyrosine levels in carrot and tobacco tissues indicating that there was no feedback control of the last step in phenylalanine and tyrosine biosynthesis.     

Enzymes of the Tryptophan Pathway in Three Bacillus Species

The tryptophan synthetic pathway was characterized in three species of Bacillus, B. subtilis, B. pumilus, and B. alvei. They share the common features of a pathway which is subject to tryptophan repression, contains no unexpected complexes among the five enzymes, exhibits dissociable anthranilate synthase enzymes which do not require phosphoribosyl transferase for amidetransfer activity, contains separate indoleglycerol phosphate synthase and phosphoribosylanthranilate isomerase enzymes, and contains similar tryptophan synthetase multimers. In looking at these characteristics in detail however, differences among the three species became apparent, as, for example, in the complementation observed between the alpha and beta(2) components of tryptophan synthetase, and the dissociation patterns of the large and small components of anthranilate synthase. The results demonstrate some pitfalls in attempting to compare multimeric enzymes in crude extracts from different organisms.     

Documentation of Auxotrophic Mutation in Blue-Green Bacteria: Characterization of a Tryptophan Auxotroph in Agmenellum quadruplicatum

A tryptophan-requiring auxotroph of Agmenellum quadruplicatum strain BG1, a species of blue-green bacteria, was isolated by means of a nitrosoguanidine-penicillin procedure. Its growth characteristics were determined, and the enzymological block was identified in the A activity of tryptophan synthetase. Starvation of the auxotroph for tryptophan resulted in the derepression of the synthesis of all five enzymes. The first four enzymes derepressed 2- to 3-fold, and tryptophan synthetase B derepressed 20-fold. In the parental prototroph, BG1, anthranilate synthetase was active in crude extracts with ammonia as the amino donor reactant, but not with glutamine.     

Tryptophan catabolism in Bacillus megaterium.

Bacillus megaterium grows in a medium containing L-tryptophan as the sole carbon, nitrogen, and energy source. Kynurenine, anthranilic acid, and catechol are metabolic intermediates, suggesting that this organism used the anthranilic acid pathway for tryptophan degradation. Cells that grow on L-tryptophan oxidize kynurenine, alanine, and anthranilic acid and the presence of tryptophan oxygenase (EC 1.13.1.12), kynureninase (EC 3.7.1.3), and catechol oxygenase (EC 1.13.1.1) in cell extracts provide additional evidence for the degradative pathway in B. megaterium. Tryptophan oxygenase is inhibited by sodium azide, potassium cyanide, and hydroxylamine, indicating that the enzyme has a functional heme group. D-Tryptophan is not a substrate for tryptophan oxygenase, and the D-isomer does not inhibit this enzyme. Formamidase (EC 3.5.1.9) and anthranilate hydroxylase are not detectable in extracts. Tryptophan catabolism is inducible in B megaterium and is subject to catabolite repression by glucose and glutamate. Arginine does not cause repression, and kynurenine induces both tryptophan oxygenase and kynureninase.     

Studies on the subunits of the anthranilate synthetase-phosphoribosyltransferase enzyme complex from Salmonella typhimurium.

Both uncomplexed subunits of the anthranilate synthetase-phosphoribosyltransferase enzyme complex from Salmonella typhimurium have an absolute requirement for divalent metal ions which can be satisfied by Mg²⁺, Mn²⁺, or Co²⁺. The metal ion kinetics for uncomplexed anthranilate synthetase give biphasic double-reciprocal plots and higher apparent K_m values than those for anthranilate synthetase in the enzyme complex. In contrast, the apparent K_m values for phosphoribosyltransferase are the same whether the enzyme is uncomplexed or complexed with anthranilate synthetase. This suggests that the metal ion sites on anthranilate synthetase, but not those on phosphoribosyltransferase, are altered upon formation of the enzyme complex. These results and the results of studies reported by others, suggest that complex formation between anthranilate synthetase and phosphoribosyltransferase leads to marked alterations at the active site of the former, but not the latter enzyme. Uncomplexed anthranilate synthetase can be stoichiometrically labeled with Co(III) under conditions which lead to inactivation of 75% of its activity. A comparison of the effects of anthranilate and tryptophan on phosphoribosyltransferase activity in the uncomplexed and complexed forms shows that anthranilate, but not tryptophan, inhibits the uncomplexed enzyme. The complexed phosphoribosyltransferase shows substrate inhibition by anthranilate binding to the phosphoribosyltransferase subunits. In contrast, in a tryptophan-hypersensitive variant complex, anthranilate inhibits phosphoribosyltransferase activity by acting on the anthranilate synthetase subunits. The data are interpreted to mean that there are two classes of binding sites for anthranilate, one on each type of subunit, which may participate in the regulation of anthranilate synthetase and phosphoribosyltransferase under different conditions.     

Tryptophan Metabolism in Ochromonas malhamensis

Tryptophan enhanced the growth of Ochromonas malhamensis at concentrations up to 0.4 mg/ml; higher concentrations inhibited, the growth inhibition being reversible by tyrosine and adenine. The presence of a tryptophan synthetase system in vitro was demonstrated. Tyrosine and phenylalanine stimulated the activity of this enzyme. The uptake of exogenous tryptophan was accompanied by an increase in the free tryptophan pool which in turn suppressed the tryptophan synthetase system, thus pointing to a controlled mechanism. Incorporation of tryptophan in the growth medium enhanced the biosynthesis of folate-active compounds. An elucidation of the mode of action of tryptophan is attempted on the basis of known metabolic pathways.     

Regulation of the chorismic acid branch-point in aromatic acid synthesis in blue-green and green algae

Summary In extension of previous studies on the regulation of the aromatic amino acid pathway in blue-green and green algae the control of two branch-point enzymes, namely chorismate mutase and anthranilate synthetase has been studied. The activity of chorismate mutase in these organisms is effectively inhibited by l-tyrosine or l-phenylalanine. l-tryptophan, in contrast, proved to be a positive effector of the enzyme: in the absence of phenylalanine or tyrosine tryptophan slightly stimulated chorismate mutase activity; this stimulation was even brought about in the presence of excess phenylalanine or tyrosine, irrespective if the enzyme had been preincubated with these inhibitors or not. Tryptophan thus proved to completely revert the feedback inhibition of this enzyme by phenylalanine or tyrosine. Substrate saturation curves of chorismate mutase activity are hyperbolic in the presence of tryptophan and sigmoid in the presence of phenylalanine or tyrosine. In contrast to the enzymes of the green algae investigated, chorismate mutase activity of Anacystis nidulans, a member of the class of the blue-green algae was not affected by any of the aromatic amino acids.The activity of anthranilate synthetase, the second enzyme of the chorismic acid branch-point of the pathway was consistently inhibited by l-tryptophan in all the organisms tested. The results described here bear significance on the regulation of a multi-branched pathway the first enzyme of which is inhibited just by one endproduct.     

Regulation of the tryptophan synthetic enzymes in Clostridium butyricum

Experiments concerned with the regulation of the tryptophan synthetic enzymes in anaerobes were carried out with a strain of Clostridium butyricum. Enzyme activities for four of the five synthetic reactions were readily detected in wild-type cells grown in minimal medium. The enzymes mediating reactions 3, 4, and 5 were derepressed 4- to 20-fold, and the data suggest that these enzymes are coordinately controlled in this anaerobe. The first enzyme of the pathway, anthranilate synthetase, could be derepressed approximately 90-fold under these conditions, suggesting that this enzyme is semicoordinately controlled. Mutants resistant to 5-methyl tryptophan were isolated, and two of these were selected for further analysis. Both mutants retained high constitutive levels of the tryptophan synthetic enzymes even in the presence of repressing concentrations of tryptophan. The anthranilate synthetase from one mutant was more sensitive to feedback inhibition by tryptophan than the enzyme from wild-type cells. The enzyme from the second mutant was comparatively resistant to feedback inhibition by tryptophan. Neither strain excreted tryptophan into the culture fluid. Tryptophan inhibits anthranilate synthetase from wild-type cells noncompetitively with respect to chorismate and uncompetitively with respect to glutamine. The Michaelis constants calculated for chorismate and glutamine are 7.6 x 10(-5)m and 6.7 x 10(-5)m, respectively. The molecular weights of the enzymes estimated by zonal centrifugation in sucrose and by gel filtration ranged from 24,000 to 89,000. With the possible exception of a tryptophan synthetase complex, there was no evidence for the existence of other enzyme aggregates. The data indicate that tryptophan synthesis is regulated by repression control of the relevant enzymes and by feedback inhibition of anthranilate synthetase. That this enzyme system more closely resembles that found in Bacillus than that found in enteric bacteria is discussed.     

Heterocyst differentiation and tryptophan metabolism in the cyanobacterium Anabaena sp. CA

Anabaena sp. CA does not synthesize heterocysts or express nitrogenase activity when grown with nitrate as the nitrogen source. Heterocysts and nitrogenase are induced in such cultures by various tryptophan analogs. The effect does not require inhibition of de novo protein synthesis in the culture. It is restricted to tryptophan analogs only, and, more specifically, to those which can be incorporated into proteins. dl-7-Azatryptophan was effective at triggering both heterocysts and nitrogenase when incubated in the culture for only 1–2 h, even though 6–7 h was required for heterocysts to fully mature and nitrogenase activity to be expressed. Chloramphenicol completely negated this effect, supporting the idea that the analogs are either incorporated into protein themselves or trigger the synthesis of proteins which initiate complete development of mature heterocysts. Using toluene-permeabilized cells, we have shown that anthranilate synthetase, the first key enzyme in tryptophan biosynthesis, has glutamine-dependent activity. This activity can be effectively feedback inhibited by the various tryptophan analogs at concentrations which are also effective in triggering heterocyst differentiation. These data provide firm evidence for a link between tryptophan biosynthesis, nitrogenase synthesis, heterocyst differentiation, and primary ammonia assimilation.     

Study of a corn (Zea mays L.) mutant (blue fluorescent-1) which accumulates anthranilic acid and its β-glucoside

A corn (Zea mays L.) mutant, blue fluorescent-1 (bf), is described that shows ultraviolet light induced blue fluorescence in young seedling leaves if homozygous for the mutant gene, and in anthers if either homozygous or heterozygous. The blue fluorescent compounds were extracted with acetone and separated by paper chromatography. Anthranilic acid was present and the beta-glucoside was also identified by paper chromatography and beta-glucosdase and acid treatment. A third major fluorescent compound was not identified, but it was convertible to anthranilic acid by acid treatment. Anthranilate synthetase from mutant plants was 3-40 times more active and was also more resistant to feedback inhibition by tryptophan than was the enzyme from normal plants. The high activity and feedback resistance would both lead to anthranilate accumulation. Anthranilate-phosphoribosylpyrophosphate phosphoribosyltransferase (PR transferase), the enzyme which usually utilizes anthranilate in the tryptophan pathway, was inhibited by the beta-glucoside of anthranilic acid in a noncompetitive manner and showed very little activity in the mutant plant extract. This inhibition of the enzyme which utilizes anthranilate would also lead to accumulation. Apparently the oversynthesis of anthranilate leads to the formation of the beta-glucoside, which inhibits anthranilate utilization. The fluorescent compounds are absent in seed, but form on germination. The levels decrease with age after 35 days postgermination, but are still present in leaves during grain filling.     

Regulation of Tryptophan Biosynthetic Enzymes in Neurospora crassa

The formation of enzymatic activities involved in the biosynthesis of tryptophan in Neurospora crassa was examined under various conditions in several strains. With growth-limiting tryptophan, the formation of four enzymatic activities, anthranilic acid synthetase (AAS), anthranilate-5-phosphoribosylpyrophosphate phosphoribosyl transferase (PRAT), indoleglycerol phosphate synthetase (InGPS), and tryptophan synthetase (TS) did not occur coordinately. AAS and TS activities began to increase immediately, whereas PRAT and InGPS activities began to increase only after 6 to 12 hr of incubation. In the presence of amitrole (3-amino-1,2,4-triazole), the formation of TS activity in a wild-type strain was more greatly enhanced than were AAS and InGPS activities. With a tr-3 mutant, which ordinarily exhibits an elevated TS activity, amitrole did not produce an increase in TS activity greater than that observed on limiting tryptophan. With tr-3 mutants, the increased levels of TS activity could be correlated with the accumulation of indoleglycerol in the medium; prior genetic blocks which prevented or reduced the synthesis of indoleglycerol also reduced the formation of TS activity. The addition of indoleglycerol to cultures of a double mutant (tr-1, tr-3) which could not synthesize indoleglycerol markedly stimulated the production of TS activity but not PRAT activity; the production of TS activity reached the same level with limiting or with excess tryptophan. A model explaining these and other related observations on enzyme formation in N. crassa is proposed.