Inducible and constitutive kynureninases. Control of the inducible enzyme activity by transamination and inhibition of the constitutive enzyme by 3-hydroxyanthranilate.

The inducible kynureninase from Neurospora crassa is inactivated by incubation with L-alanine or L-ornithine. The inactivated enzyme is resolved to the apoenzyme by dialysis. Reactivation of the apoenzyme is achieved by incubation with pyridoxamine 5'-phosphate plus pyruvate, as well as with pyridoxal 5'-phosphate. The kynurenine hydrolysis proceeds linearly in the presence of added pyridoxal 5'-phosphate, or pyridoxamine 5'-phosphate plus pyruvate. These findings indicate that the fungal inducible kynureninase can act as an amino-transferase to control the enzyme activity, and that the control mechanism is similar to that reported for the bacterial kynureninase (Moriguchi, M. & Soda, K. (1973) Biochemistry 12, 2974-2980). The ratio of kynureninase activity to aminotransferase activity was determined with bacterial and fungal enzymes. All the inducible kynureninases from various fungal species examined are also controlled by the transamination. In contrast, the pig liver kynureninase and the fungal constitutive enzymes are little or not at all affected by preincubation with amino acids. Thus, the present regulatory mechanism does not operate in these constitutive-type enzymes. The rate of hydrolysis of L-3-hydroxykynurenine by the pig liver enzyme decreases with increase in the incubation time; the enzyme is inhibited by 3-hydroxyanthranilate produced from L-3-hydroxykynurenine. The inhibition is found in all the constitutive-type enzymes, suggesting that 3-hydroxyanthranilate plays a regulatory role in NAD biosynthesis from tryptophan.     

Distinct kynureninase and hydroxykynureninase activities in microorganisms: occurrence and properties of a single physiologically discrete enzyme in yeast

(i) Saccharomyces cerevisiae grown in the presence of 1.0 mM l-tryptophan slowly excreted fluorescent material that was chromatographically identifiable as 3-hydroxyanthranilate but did not excrete detectable amounts of anthranilate nor rapidly deplete the medium of l-tryptophan. Under similar growth conditions, Neurospora crassa rapidly excretes anthranilate and rapidly depletes the medium of l-tryptophan. (ii) Chromatographic analysis of crude extracts from yeast revealed a single kynureninase-type enzyme whose synthesis was not measurably affected by the presence of tryptophan in the medium. Previous studies have provided evidence for two kynureninase-type enzymes in N. crassa, an inducible kynureninase and a constitutive hydroxykynureninase. (iii) Kinetic analysis of the partially purified yeast enzyme provided Michaelis constants for l-3-hydroxykynurenine and l-kynurenine of 6.7 x 10(-6) and 5.4 x 10(-4) M, respectively. This and other kinetic properties of the yeast enzyme are comparable to those reported for the constitutive enzyme from N. crassa. (iv) These findings suggest that S. cerevisiae has in common with N. crassa the biosynthetic enzyme hydroxykynureninase but lacks the catabolic enzyme kynureninase. Therefore, it can be predicted that, unlike N. crassa, S. cerevisiae does not carry out the tryptophan-anthranilate cycle. Distinct kynureninase-type enzymes may exist in other microorganisms and in mammals.     

Evidence for distinct kynureninase and hydroxykynureninase activities in Neurospora crassa

Previous studies have indicated that a single enzyme, "kynureninase," catalyzes the reactions of l-kynurenine to anthranilate and l-3-hydroxykynurenine to 3-hydroxyanthranilate in Neurospora crassa and in other organisms. The present report describes separate enzymes which catalyze these reactions in N. crassa. The first, a kynureninase, preferentially catalyzes kynurenine to anthranilate and is induced over 400-fold by tryptophan or a catabolite of tryptophan. The second, a hydroxykynureninase, is constitutive or noninducible by tryptophan and preferentially catalyzes l-3-hydroxykynurenine to 3-hydroxyanthranilate. The physiological significance of these enzymes may be inferred from the facts that (i) the noninducible enzyme hydroxykynureninase appears to be the main enzyme present in uninduced cells that is capable of catalyzing l-3-hydroxykynurenine to 3-hydroxyanthranilate for the indispensible synthesis of nicotinamide adenine dinucleotide, and (ii) the inducible enzyme kynureninase is induced by tryptophan to a concentration far in excess of that needed to meet the requirements of the cells for nicotinamide adenine dinucleotide, resulting in the excretion of anthranilate into the medium.     

Isolation and Characterization of Low-Kynureninase Mutants of Neurospora crassa

Two kynureninase activities are known in Neurospora crassa, one of which (kynureninase I) is inducible, the other (kynureninase II) being constitutive. A method is described for the isolation of low-kynureninase mutants of N. crassa. When grown on an inducer, the mutants show significantly less kynureninase I activity compared with wild type, whereas constitutive kynureninase II activity is unaffected. Since a low level of kynureninase I activity remains in the mutants examined, the mutations may be in a regulatory gene or genes. Other experiments are described concerning the molecular weights of the two enzymes and the intracellular localization and specificity of kynureninase II.     

Regulation of two nickel-requiring (inducible and constitutive) hydrogenases and their coupling to nitrogenase in Methylosinus trichosporium OB3b.

Two uptake hydrogenases were found in the obligate methanotroph Methylosinus trichosporium OB3b; one was constitutive, and a second was induced by H2. Both hydrogenases could be assayed by measuring methylene blue reduction anaerobically or by coupling their activity to nitrogenase acetylene reduction activity in vivo in an O2-dependent reaction. The H2 concentration for half-maximal activity of the inducible and constitutive hydrogenases in both assays was 0.01 and 0.5 bar (1 and 50 kPa), respectively, making it easy to distinguish these enzymes from one another both in vivo and in vitro. Hydrogen uptake was shown to be coupled to ATP synthesis in methane-starved cells. Methane, methanol, formate, succinate, and glucose all repressed the H2-mediated synthesis of the inducible hydrogenase. Furthermore, this enzyme was only expressed in N-starved cultures and was repressed by NH4+ and NO3-; synthesis of the constitutive hydrogenase was not affected by excess N in the growth medium. In nickel-free, EDTA-containing medium, the activities of these two enzymes were negligible; however, both enzyme activities appeared rapidly following the addition of nickel to the culture. Chloramphenicol, when added along with nickel, had no effect on the rapid appearance of either the constitutive or inducible activity, indicating that nickel is not required for synthesis of the hydrogenase apoproteins. These observations all suggest that these hydrogenases are nickel-containing enzymes. Finally, both hydrogenases were soluble and could be fractionated by 20% ammonium sulfate; the constitutive enzyme remained in the supernatant solution, while the inducible enzyme was precipitated under these conditions.     

Crystal structure of Homo sapiens kynureninase

Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.     

Alteration of Tryptophan-mediated Regulation in Neurospora crassa by Indoleglycerol Phosphate

The accumulation of imidazoleglycerol phosphate during growth of Neurospora crassa in the presence of 3-amino-1,2,4-triazole was found to cause derepression of tryptophan synthetase and to inhibit the induction of kynureninase. Accumulation of indoleglycerol phosphate in response to growth in the presence of indole acrylic acid or anthranilic acid was also accompanied by derepressed synthesis of tryptophan synthetase. Enzyme synthesis in mutants (his-7 and trp-4) unable to form these intermediates was not altered under similar conditions. The rate of formation of tryptophan synthetase and kynureninase was found to differ in the presence of tryptophan and indole.     

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.     

A comparative study on the inhibition of human and bacterial kynureninase by novel bicyclic kynurenine analogues

A series of novel bicyclic analogues of kynurenine were synthesised as inhibitors of kynureninase. The tryptophan-induced bacterial enzyme from Pseudomonas. fluorescens was compared to the constitutive recombinant human enzyme expressed in a baculovirus/insect cell system, with regard to their inhibition by these compounds. All the compounds studied were found to be simple competitive, reversible inhibitors of kynureninase. It was found that altering the size of the second ring of the inhibitor affected the observed Ki values for both enzymes. The addition of an oxygen atom into the second ring had little effect on binding to the bacterial enzyme but gave a more potent inhibitor of human kynureninase. Of the compounds tested, a naphthyl analogue of desaminokynurenine was found to be the most potent inhibitor for both enzymes with Ki values of 5 and 22 microM for bacterial and human enzyme respectively. This report also describes an alternative system for the expression of recombinant human kynureninase which is more convenient for expression in mammalian cells and produces a relatively greater quantity of enzyme.     

Isolation and Properties of Phenol Utilizing Microorganisms with Special Reference to Catechol 1,2-Oxygenase

Phenol utilizing yeasts were isolated from soil. The relationship were examined between distribution of phenol uptake rate using intact cells and distribution of the activities of catechol 1,2-oxygenase which is one of the key enzymes in phenol metabolism. Two of the isolates showed catechol 1,2-oxygenase activity even when grown in glucose medium, though the enzyme activity was about 1% of the full activity induced by phenol. Partially constitutive mutants for catechol 1,2-oxygenase were obtained by mutagenesis of an inducible strain. The level of mutant enzyme activity was close to that of the isolated constitutive strain. One isolate, Trichosporon cutaneum, preferentially utilized phenol to glucose in medium containing both phenol (200 ppm) and glucose (0.1%), until the concentration of phenol decreased to 10–20 ppm.     

Tryptophan Metabolism and Kynureninase Induction in Mutants of Neurospora crassa Resistant to 4-Methyl-Tryptophan

Mutants of Neurospora crassa that are resistant to 4-methyl-tryptophan were found to differ in ability to synthesize kynureninase in the presence of the inducers kynurenine, 3-OH-kynurenine, N-formyl-kynurenine, tryptophan, and indole. One strain (mtr26), although incapable of accumulating intracellular pools of these compounds, showed induced synthesis of kynureninase, whereas the second (mtr21) could neither accumulate nor be induced by them. Strain mtr21, with the suppressor su(mtr), could not be induced by indole but was induced by tryptophan and kynurenine derivatives. These results suggest that the mtr mutation, in addition to altering the ability of these strains to concentrate tryptophan and its metabolites, may have some effect on either the intracellular distribution of tryptophan or directly on the synthesis of kynureninase.     

Enzyme activities involved in tryptophan metabolism along the kynurenine pathway in rabbits

The following enzyme activities of the tryptophan-nicotinic acid pathway were studied in male New Zealand rabbits: liver tryptophan 2,3-dioxygenase, intestine indole 2,3-dioxygenase, liver and kidney kynurenine 3-monooxygenase, kynureninase, kynurenine-oxoglutarate transaminase, 3-hydroxyanthranilate 3,4-dioxygenase, and aminocarboxymuconate-semialdehyde decarboxylase. Intestine superoxide dismutase and serum tryptophan were also determined. Liver tryptophan 2,3-dioxygenase exists only as holoenzyme, but intestine indole 2,3-dioxygenase is very active and can be considered the key enzyme which determines how much tryptophan enters the kynurenine pathway also under physiological conditions. The elevated activity of indole 2,3-dioxygenase in the rabbit intestine could be related to the low activity of superoxide dismutase found in intestine. Kynurenine 3-monooxygenase appeared more active than kynurenine-oxoglutarate transaminase and kynureninase, suggesting that perhaps a major portion of kynurenine available from tryptophan may be metabolized to give 3-hydroxyanthranilic acid, the precursor of nicotinic acid. In fact, 3-hydroxyanthranilate 3,4-dioxygenase is much more active than the other previous enzymes of the kynurenine pathway. In the rabbit liver 3-hydroxyanthranilate 3,4-dioxygenase and aminocarboxymuconate-semialdehyde decarboxylase show similar activities, but in the kidney 3-hydroxyanthranilate 3,4-dioxygenase activity is almost double. These data suggest that in rabbit tryptophan is mainly metabolized along the kynurenine pathway. Therefore, the rabbit can also be a suitable model for studying tryptophan metabolism in pathological conditions.     

Regulation of pyruvate kinases from Fusarium oxysporum.

Two types of pyruvate kinases were found in Fusarium oxysporum. One type (inducible) was present mainly during the early stages of growth on glucose or sucrose and displayed Michaelis-Menten kinetics with respect to phosphoenolpyruvate and adenosine diphosphate. The major type (constitutive) was present under all conditions of growth and displayed in the absence of potassium ions, a sigmoidal substrate saturation curve when phosphoenolpyruvate was used as the variable substrate. In the presence of potassium ions the saturation curve for phosphoenolpyruvate exhibits a plateau at half-maximal velocity. The effects of various metabolites on the activity of the inducible and constitutive kinases were also studied. Fructose-1,6-diphosphate, cyclic AMP, acetyl CoA, tryptophan, and phenylalanine had no effect on the activity of the enzymes. Citrate was a potent inhibitor of the constitutive pyruvate kinase activity and increased the sigmoidicity of the saturation curve for phosphoenolpyruvic acid. In the presence of K+, the bimodal plot observed in the absence of citrate gradually changed to a hyperbolic shape as the concentration of citric acid was increased. In the presence of K+ and ADP as the variable substrate citric acid converted the hyperbolic plot to a sigmoidal one. Citrate had no effect on the inducible enzyme.     

The effect of pyridoxine deficiency on the metabolism of the aromatic amino acids by isolated rat liver cells

The total activity of three key enzymes and the flux through eight steps of aromatic amino acid metabolism have been determined in liver cells isolated from rats fed either control or pyridoxine-free diet for 5-6 weeks. The pyridoxine-free diet caused a decrease in the catabolism of tyrosine and phenylalanine because of a drop in the flux through tyrosine aminotransferase. This decrease of expressed cellular tyrosine aminotransferase activity can be fully explained in terms of loss of cofactor. Larger decreases in the catabolism of tryptophan were seen after pyridoxine deprivation. The decreased extent of tryptophan catabolism can be solely attributed to loss of cofactor or increased degradation of kynureninase. Inhibition of tryptophan 2,3-dioxygenase was seen in pyridoxine deficiency, probably because of the buildup of the kynurenine metabolites. The control strength of kynureninase, for flux through kynureninase, was calculated to be less than or equal to 0.004, but 0.41 after pyridoxine deprivation. The sensitivity of the three pathways to pyridoxine deprivation is interpreted and discussed in terms of the different affinities for pyridoxal phosphate and the control strengths of the pyridoxal phosphate-dependent enzymes, tyrosine aminotransferase and kynureninase.     

Enzyme activities along the tryptophan-nicotinic acid pathway in alloxan diabetic rabbits

Recent data from our laboratory have indicated that the rabbit is a suitable animal model for the study of enzyme activities of the tryptophan-nicotinic acid pathway. We report here the pattern of tryptophan metabolism in rabbits made diabetic with alloxan treatment, and hypercholesterolemic with a high-cholesterol diet. A group of rabbits with only hypercholesterolemia was also considered. The enzymes assayed were: liver tryptophan 2,3-dioxygenase (TDO), intestine indoleamine 2,3-dioxygenase (IDO), liver and kidney kynurenine 3-monooxygenase, kynurenine-oxoglutarate transaminase, kynureninase, 3-hydroxyanthranilate 3,4-dioxygenase and aminocarboxymuconate-semialdehyde decarboxylase.TDO showed a reduction of specific activity in liver of diabetic-hyperlipidemic and hyperlipidemic rabbits compared to controls. Intestine IDO activities and liver and kidney kynurenine monooxygenase were unchanged with respect to controls.Kynurenine-oxoglutarate transaminase and kynureninase activities were reduced in the kidneys, but not in the liver, of diabetic-hyperlipidemic rabbits.The main finding was the reduction of 3-hydroxyanthranilate 3,4-dioxygenase activity (expressed as activity per g of fresh tissue) in the liver and kidneys of diabetic-hypercholesterolemic and hyperlipidemic rabbits compared to controls. Conversely, aminocarboxymuconate-semialdehyde decarboxylase activity was significantly higher in diabetic hypercholesterolemic rabbits in comparison with control and hypercholesterolemic rabbits.These data demonstrate that also in diabetic rabbits there is an alteration of tryptophan metabolism at the level of 3-hydroxyanthranilic acid-->nicotinic acid step. Also dyslipidemia seems to be involved in enzyme activity variations of the tryptophan metabolism along the kynurenine pathway.     

In vivo regulation of intermediate reactions in the pathway of tryptophan biosynthesis in Neurospora crassa

The in vivo regulation of intermediate reactions in the pathway of tryptophan synthesis in Neurospora crassa was examined in a double mutant (tr-2, tr-3) which lacks the functions of the first and last enzymes in the pathway from chorismic acid to tryptophan. The double mutant can convert anthranilic acid to indole and indole-3-glycerol, and the production of these indolyl compounds by germinated conidia was used to estimate the activity of the intermediate enzymes in the pathway. Indole-synthesizing activity was maximal in germinated conidia obtained from cultures in which the levels of l-tryptophan were growth-limiting; the formation of this activity was markedly repressed when the levels of l-tryptophan exceeded those required for maximal growth. d-, 5-methyl-dl-, and 6-methyl-dl-tryptophan were less effective than l-tryptophan, and 4-methyl-dl-tryptophan, tryptamine, and indole-3-acetic acid were ineffective in repressing the formation of indole-synthesizing activity; anthranilic acid stimulated the formation of indole-synthesizing activity. Preformed indole-synthesizing activity was strongly and specifically inhibited by low levels of l-tryptophan; several related compounds were ineffective as inhibitors. These results suggest that, in addition to repression, an end product feedback inhibition mechanism is operative on an intermediate enzyme(s) in tryptophan biosynthesis. The relation of these results to other in vivo and in vitro studies and to general aspects of the regulation of tryptophan biosynthesis in N. crassa are discussed.     

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.     

Lifetime of bacterial messenger ribonucleic acid

Moses, V. (University of California, Berkeley), and M. Calvin. Lifetime of bacterial messenger ribonucleic acid. J. Bacteriol. 90:1205-1217. 1965.-When cells from a stationary culture of Escherichia coli were placed in fresh medium containing inducer for beta-galactosidase, growth, as represented by increase in turbidity and by total protein synthesis, started within 30 sec. By contrast, beta-galactosidase synthesis was greatly delayed compared with induction during exponential growth. Two other inducible enzymes (d-serine deaminase and l-tryptophanase) and one repressible enzyme (alkaline phosphatase) showed similar lags. The lags were not due to catabolite repression. They could not be reduced by pretreatment of the culture with inducer, or by supplementing the fresh medium with amino acids or nucleotides. The lag was also demonstrated by an i(-) mutant constitutive for beta-galactosidase synthesis. An inhibitor of ribonucleic acid (RNA) synthesis, 6-azauracil, preferentially inhibited beta-galactosidase synthesis compared with growth in both inducible and constitutive strains. Puromycin, an inhibitor of protein synthesis, acted as an inhibitor at additional sites during the induction of beta-galactosidase synthesis. No inhibition of the reactions proceeding during the first 20 sec of induction was observed, but puromycin seemed to prevent the accumulation of messenger RNA during the period between 20 sec and the first appearance of enzyme activity after 3 min. It is suggested that these observations, together with many reports in the literature that inducible enzyme synthesis is more sensitive than total growth to some inhibitors and adverse growth conditions, can be explained by supposing that messenger RNA for normally inducible enzymes is biologically more labile than that for some normally constitutive proteins. The possible implications of this hypothesis for the achievement of cell differentiation by genetic regulation of enzyme synthesis are briefly discussed.     

Histidine-Mediated Control of Tryptophan Biosynthetic Enzymes in Neurospora crassa

The formation of the five tryptophan biosynthetic enzymes of Neurospora crassa was shown to be derepressed in histidine-starved cells. This histidine-mediated derepression was not due to a lowered intracellular concentration of tryptophan in these cells. Furthermore, histidine-mediated derepression of tryptophan enzymes was found to be coordinate and not subject to reversal by tryptophan of either exogenous or biosynthetic origin. The synthesis of tryptophan enzymes also was found to be coordinate in cells which were not histidine-starved. Although histidine is clearly involved in regulating the synthesis of tryptophan enzymes, it did not prevent either tryptophan-mediated derepression of tryptophan enzymes or indole-3-glycerol phosphate-mediated derepression of tryptophan synthetase.     

Tryptophan catabolism during sporulation in Bacillus cereus

1. Two intermediates of tryptophan catabolism were isolated from a sporulating culture of Bacillus cereus and identified as anthranilic acid and kynurenine by their spectral properties. 2. During sporulation the rate of formation of anthranilic acid and kynurenine by whole cells increased and reached a maximum at the pre-spore stage. 3. The specific activities of tryptophan pyrrolase and formylase also increased during sporulation and exhibited a maximal activity at the pre-spore stage. 4. Kynureninase activity reached a maximum during early stages of sporulation and then started to decline. 5. There was a net increase in the activity of tryptophan pyrrolase when cells were grown in the presence of l-tryptophan or dl-kynurenine. 6. The cultures exhibited the maximal activity of kynureninase 2h earlier in the presence of dl-kynurenine whereas l-tryptophan delayed the appearance of the maximal activity by 2h. 7. The omission of glucose from the medium had no effect on the pattern of development of tryptophan pyrrolase during growth and sporulation. 8. On the addition of tryptophan to a chemically defined medium no significant change in the pattern of development of tryptophan pyrrolase was observed.