Indoleglycerol Phosphate Research Articles

Limited proteolysis of N-(5′-phosphoribosyl)anthranilate isomerase: Indoleglycerol phosphate synthase from Escherichia coli yields two different enzymically active, functional domains

The native enzyme must be denatured either by sodium dodecyl sulfate or by urea before limited proteolysis can occur. Under these conditions only one or two peptide bonds are hydrolyzed by each of the following proteases: Staphylococcal V8 protease, trypsin and elastase. The amino-terminal amino acid sequences were determined to identify the cleavage sites. The new sequences comprise approximately 20% of the entire polypeptide chain, and show good agreement with the nucleotide sequence of the trpC gene. Both V8 protease † † Abbreviations used: V8 protease, extracellular protease from Staphylococcus aureus strain V8: PRA, N-(5′-phosphoribosyl)anthranilate; InGP, indoleglycerol phosphate; CdRP, 1-(2-carboxyphenyl-amino)-1-deoxyribulose 5-phosphate; SDS, sodium dodeoyl sulfate. and elastase yield large carboxy-terminal fragments, about two thirds of the size of the parent enzyme, and corresponding small amino-terminal fragments. Trypsin cleaves a single peptide bond in the last one third of the polypeptide chain. After separation of the fragments, removal of dodecyl sulfate and renaturation, only the large fragments fold to stable structures. The small fragments precipitate. The large amino-terminal fragment catalyzes only the synthesis of indoleglycerol phosphate and precipitates when solutions are frozen and thawed. The large carboxy-terminal fragment catalyzes only the isomerization of N-(5′-phosphoribosyl)anthranilate and is stable towards freezing and thawing. These studies prove that the intact bifunctional enzyme consists of two autonomously folding, functional domains. They also support the notion that the bifunctional enzyme may have arisen by the fusion of separate ancestral genes, and that stabilization of the intrinsically labile indoleglycerol phosphate synthase domain by interdomain interactions is functionally advantageous.

Purification and characterization of histidyl-transfer RNA synthetase from Neurospora crassa

Histidyl-tRNA synthetase ( l-histidine:tRNA His ligase (AMP-forming), EC 6.1.1.21) has been purified 921-fold from crude extracts of lyophilized mycelia of Neurospora crassa. Sodium dodecyl sulfate gel electrophoresis at pH 8.9 of the purified enzyme yields one band with an apparent M r of 62 500. The estimated M r by Sephadex gel filtration is 125 000. Thus the native histidyl-tRNA synthetase of N. crassa is a dimer, composed of two identical subunits. The K m values determined in the enzyme-catalyzed esterification of [ 14C]-histidine to tRNA His are: for histidine, 5.8·10 −6 M , for ATP, 5.9·10 −4 M , and for tRNA His, 1.2·10 −7 M. Effects of various intermediates of the histidine, tryptophan and arginine biosynthetic pathways on histidyl-tRNA synthetase activity were studied. The K i values for imidazoleglycerol phosphate and histidinol (histidine intermediates and competitive inhibitors of the enzyme) are 1.1·10 −2 M, 1.3·10 −6 M, respectively. The K i for indoleglycerol phosphate (a tryptophan intermediate and non-competitive inhibitor) is 1.2·10 −3 M.

Isolation and characterization of two tryptophan biosynthetic enzymes, indoleglycerol phosphate synthase and phosphoribosyl anthranilate isomerase, from Bacillus subtilis.

Two of the enzymes responsible for tryptophan biosynthesis in Bacillus subtilis have been extensively purified. These proteins are indole-3-glycerol phosphate synthase and N-(5'-phosphoribosyl) anthranilate isomerase. By comparison to the non-differentiating enteric bacteria in which these two enzymes are fused into a single polypeptide, the isolation of the indoleglycerol phosphate synthase and phosphoribosyl anthranilate isomerase from B. subtilis has demonstrated that the two proteins are separate species in this organism. The two enzymes were clearly separable by anion-exchange chromatography without any significant loss of activity. Molecular weights were determined for both enzymes by gel filtration and sodium dodecyl sulfate-slab gel electrophoresis, and indicated that the indoleglycerol phosphate synthase is the slightly larger of the two proteins. The minimum molecular weight for indoleglycerol phosphate synthase was 23,500, and that for phosphoribosyl anthranilate isomerase was 21,800. Both enzymes have been examined as to conditions necessary to achieve maximal activity of their individual functions and to maintain that activity.

Affinity Chromatography of Tryptophan Synthase fromEscherichia coli

Inhibition studies and affinity chromatography indicate that derivatives of tryptophanol phosphate are suitable ligands for the affinity chromatography of tryptophan synthase. A phenyl group on the spacer arm strengthens the interaction of immobilized tryptophanol phosphate with the enzyme. The alpha 2 beta 2 complex specifically requires the presence of 0.3--0.5 M phosphate ions for binding. The alpha subunit binds in dilute Tris buffer, but its binding is also enhanced by the presence of phosphate ions. The beta 2 subunit binds unspecifically but strongly to the affinity material and to a variety of other immobilized hydrophobic ligands. Binding studies with suspensions of affinity material show that the alpha subunit interacts rapidly and reversibly. Indoleglycerol phosphate and indolepropanol phosphate release bound alpha 2 beta 2 complex and alpha subunit in a competitive manner, indicating that the interaction occurs biospecifically, i.e. via the active site of alpha subunit. L-Serine is a non-competitive inhibitor of binding. These results are discussed with regard to the composite-active-site hypothesis [T. E. Creighton (1970) Eur. J. Biochem, 13, 1--10]. Both the alpha subunit and the alpha 2 beta 2 complex of tryptophan synthase from Escherichia coli can be obtained with high yields and in homogenous form by absorption to the affinity material from partially purified preparations. Elution is achieved with linear gradients either of indolepropanol phosphate or of indoleglycerol phosphate or, in the case of the complex, of L-serine. At the low concentrations of the complex found in crude extracts of wild-type E. coli cells, the unexpectedly high affinity of the beta 2 subunit for hydrophobic ligands leads to partial dissociation of the complex.

Stereochemistry and mechanism of reactions catalyzed by tryptophan synthetase and its beta2 subunit

The synthesis of tryptophan from serine and indole or indoleglycerol phosphate catalyzed by native tryptophan synthetase or PZ protein is shown to proceed stereospecifically with retention of configuration at Cg. In the a$ elimination reaction of serine to give pyruvate and ammonia catalyzed by the fiz protein, the hydrogen from C, is transferred intramolecularly and without exchange with solvent protons to Cg, where it replaces the OH group with net retention of configuration. In a competing reaction, an abortive transamination in the presence of mercaptoethanol to give pyridoxamine and S-pyruvyl mercaptoethanol, the same proton is transferred to the extent of 70% to C-4’ of the cofactor when the reaction is carried out in DzO. Together with the finding of Dunathan and Voet (Dunathan, H. G. and Voet, J. G. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 3888) that the cofactor is protonated from the si face, these data fully define the geometry of the substrate l coenzyme complex and the position of an essential base relative to it. Since no isotope effect was observed in the protonation of C, of tryptophan synthesized from indole and serine in 50% DzO, the base must be monoprotic. The known inability of the enzyme to degrade tryptophan by a$ elimination is not due to inability to remove H, of tryptophan; native enzyme and j3~ protein catalyze a hydrogen exchange of tryptophan at a rate of20% and 14%, respectively, of that of tryptophan synthesis.

Biochemical identification of a tryptophan auxotroph in Rhodospirillum rubrum

A tryptophan auxotroph of aerobically grown Rodospirillum rubrum was isolated after mutagenesis and replica plating. The mutant grows on tryptophan or indole, accumulates indole glycerol phosphate, lacks the capacity to convert indole glycerol phosphate to indole glycerol phosphate to indole, and finally is defective in photosynthetic growth. The strain is, therefore, analogous to a Trp A mutant which is defective in the alpha-subunit structural gene of tryptophan synthetase.

On the mechanism of action of Escherichia coli tryptophan synthase Steady-state investigations

Tryptophan synthase from Escherichia coli (L-serine hydro-lyase (adding indole), EC 4.2.1.20) synthesizes l-tryptophan from indoleglycerol phosphate and l-serine, releasing glyceraldehyde 3-phosphate, or from indole and l-serine. The latter reaction (B reaction), catalyzed either by the β 2 species or by the (α 2β 2) complex, has been studied by steady-state methods. A sequential mechanism is indicated. Inhibition experiments with the substrate analogue benzimidazole were carried out in order to distinguish between random and ordered mechanisms. The results are compatible with a random sequential mechanism. The dissociation constants of the enzyme-substrate complexes are evaluated. When catalyzed by the tetrameric complex (α 2β 2) the B reaction is inhibited by higher concentrations of the substrate indole. This inhibition does not follow the usual substrate inhibition pattern. The question whether the binding of indole to the α-subunit exerts an inhibitory effecton the β 2 species, possibly by reversing the activation by the α subunit of the β 2 species, is discussed.

Inactivation and partial degradation of phosphoribosylanthranilate isomerase-indoleglycerol phosphate synthetase in nongrowing cultures of Escherichia coli

The stability of tryptophan biosynthetic enzyme activities was examined in cultures of repressor-negative (trpR) strains of Escherichia coli K-12 incubated under conditions of nutrient starvation of chloramphenicol inhibition. The results show that four of the five activities examined are stable under most nongrowing conditions, whereas one activity, indoleglycerol phosphate (InGP) synthetase, carried by the trpC protein, is unstable under most conditions tested. Phosphoribosylanthranilate (PRA) isomerase activity, which is also carried by the trpC protein, is unstable during starvation for ammonium, cysteine, or sulfate but is stable under other nongrowing conditions where InGP synthetase is not. InGP synthetase activity but not PRA isomerase activity is also diminished about twofold in cultures using glycerol as a carbon-energy source. These results indicate that one or both activities of the trpC protein is specifically inactivated under several culture conditions. Experiments with antibodies to the trpC protein show that sulfate-starved and ammonium-starved cultures contain 20 to 40% less immunologically reactive trpC protein than unstarved cultures. This indicates that the trpC protein is probably partially degraded under these conditions. During recovery from sulfate starvation or ammonium starvation, cultures slowly regain normal levels of InGP synthetase and PRA isomerase activities, suggesting that inactivation may be reversible.

Substrate interactions with the α-subunit of the Escherichia coli tryptophan synthase : A kinetic study of the wild-type α-subunit

The kinetics of the reversible reaction [indoleglycerol phosphate (InGP) ⇌ indole and glyceraldehyde 3-phosphate] catalyzed by the α-subunit of the Escherichia coli tryptophan synthase was examined. The forward reaction (InGP to indole) is characterized by simple Michaelis-Menton kinetics at low (<2 m m) InGP concentrations and is consistent with the binding of a single InGP molecule at the active site. At higher (>2 m m) InGP concentrations, there is a very sharp decrease in rate. This concentration-dependent rate decrease parallels a sharp concentration-dependent aggregation of InGP. This rate decrease at high InGP concentrations may be due partially to this aggregation and the consequent lowering of monomeric InGP concentration. The reverse reaction (indole to InGP) is characterized by a sigmoidal response of rate of indole concentration. The data (Hill plot and double reciprocal 1 v versus 1/[indole] 2 plot) are consistent with two strongly interacting sites, an effector site and the active site. The effect of InGP on the reverse reaction is InGP concentration dependent. At low InGP concentrations, there is an inhibition with a concomitant slight decrease in the V. Under these conditions, the two indole sites are still apparent. These data are interpreted as being due to monomeric InGP binding only to the glyceraldehyde 3-phosphate portion of the active site. At high InGP concentrations, there is an increased activation of the reverse reaction and nearly a doubling in V. In addition, the Hill slope approaches a value of 2 and the double-reciprocal plot ( 1 v versus 1/[indole] 2) is linear only at high indole concentrations. These data are consistent with the interpretation that InGP (probably the aggregate form) binds to a separate effector site and that this binding precludes the functioning of the indole effector site at low indole concentrations. The effect of 1 or 7 m m indole on the forward reaction is negligible or slightly stimulatory. At low InGP concentrations, the effect of indole may be due to a diminished activation of the active site when it is occupied by InGP rather than when it is occupied by indole and glyceraldehyde 3-phosphate. At high InGP concentrations, the effect of indole can be attributed to the displacement of InGP by indole in the InGP aggregate.

Inhibition of aminoacyl-transfer ribonucleic acid synthetases and the regulation of amino acid biosynthetic enzymes in Neurospora crassa

Growth conditions that result in the accumulation of the tryptophan intermediate indoleglycerol phosphate or of the histidine intermediate imidazoleglycerol phosphate cause mycelia of Neurospora crassa to exhibit an immediate and sustained increase in the differential rate at which the biosynthetic enzymes of the tryptophan, histidine, and arginine pathways are synthesized. These accumulated intermediates are shown to be inhibitors of the activity of aminoacyltransfer ribonucleic acid (tRNA) synthetases, as judged by an in vitro esterification assay. The tryptophan intermediate is shown to inhibit the charging of tryptophan, and the histidine intermediate is shown to inhibit charging of histidine. The inhibitions noted are consistent with the finding that the level of charged tRNATrp is decreased significantly in cells that have accumulated indoleglycerol phosphate and that of tRNAHis is decreased significantly in cells that have accumulated imidazoleglycerol phosphate. These results are interpreted as support for the involvement of aminoacyl-tRNA species in mediating cross-pathway regulation of the tryptophan, histidine, and arginine biosynthetic pathways as proposed in Lester's polyrepressor hypothesis (G. Lester, 1971). the correlations noted lead to the conclusion that Neurospora utilizes regulatory mechanisms that have the ability to react to changes in the level of charging of tRNA species.

Purification and properties of the alpha2beta2 complex of tryptophan synthetase of Proteus mirabilis.

A procedure is described for the purification of the tryptophan synthetase alpha2beta2 complex from cell extracts of Proteus mirabilis. A 30-fold purification was achieved with an overall yield of about 23% and a specific activity of 1,600. The complex can be dissociated and the subunits isolated in a pure form. The complex can be reconstituted from the isolated subunits to regain the initial activity. The alpha and beta2 subunits of the tryptophan synthetase complex of P. mirabilis are not significantly different from those of Escherichia coli and other enteric bacteria as to their physical properties, amino acid compositions, and enzymic properties. Complementation studies indicate that the alpha subunit of P. mirabilis hybridizes well with the beta2 subunit from E. coli. Similarly, the beta2 subunit of P. mirabilis readily complexes with the alpha subunits from E. coli, Salmonella typhimurium, and Serratia marcescens. The hybrids formed are all effective in catalyzing the conversion of indoleglycerol phosphate plus serine into tryptophan and glyceraldehyde 3-phosphate. However, these hybrids have reduced or no activity in the other reactions, namely, the condensation of indole and serine to form tryptophan or the aldolytic cleavage of indoleglycerol phosphate.

Regulation of enzyme synthesis in the tryptophan pathway of Acinetobacter calcoaceticus.

In Acinetobacter calcoaceticus the seven genes coding for the enzymes responsible for tryptophan synthesis map at three chromosomal locations. Two three-gene clusters, one (trpGDC) specifying the small subunit of anthranilate synthase, phosphoribosyl transferase, and indoleglycerol phosphate synthase and the other (trpFBA) specifying phosphoribosyl anthranilate isomerase and both tryptophan synthase subunits, are not linked to each other or to the trpE gene specifying the large anthranilate synthase subunit. When regulation of trp gene expression is studied in the wild type, only the level of the trpF gene product decreases upon addition of tryptophan to the medium. Tryptophan starvation of tryptophan auxotrophs, however, results in increased levels of all the tryptophan enzymes; this and additional evidence suggests that the expression of all the trp genes is subject to repression. The trpGDC genes are coordinately controlled, and the trpE gene is regulated in parallel with them. The trpFBA genes are controlled neither coordinately nor in parallel with the other trp genes, but respond proportionally when compared with each other. So far, two types of constitutive mutants have been found. The first class of mutants apparently occurs in the structural gene for a repressor protein; this repressor locus is unlinked to any of the biosynthetic trp genes and affects only the expression of trpE and the trpGDC cluster. The second class contains mutants closely linked to the trpGDC region; they overproduce only the gene products of this cluster.

The mechanism of the synthesis of indoleglycerol phosphate catalyzed by tryptophan synthase from Escherichia coli. Steady-state kinetic studies.

The mechanism of indoleglycerol phosphate synthesis from indole and D-glyceraldehyde 3-phosphate catalyzed by tryptophan synthase has been investigated by steady-state kinetic techniques. The equilibrium constant and the progress curves were measured by use of the difference in absorbance between indole and indoleglycerol phosphate. Stopped-flow measurements show that only the non-hydrated form of D-glyceraldehyde 3-phosphate serves as substrate. The product analogue indolepropanol phosphate was used as an inhibitor to discriminate between possible mechanisms. The data agree well with an ordered addition mechanism with D-glyceraldehyde 3-phosphate adding first. Mechanisms involving random addition of substrates or ordered addition with indole adding first can be excluded because indolepropanol phosphate is a competitive inhibitor only towards glyceraldehyde 3-phosphate. The high affinity of tryptophan synthase for indoleglycerol phosphate leads to product inhibition even at small extents of reaction. Glyceraldehyde 3-phosphate combines with the enzyme with an apparent second-order rate constant, which is not diffusion controlled and generates a site with high affinity for indole.

Steady‐State Kinetic Studies of the Synthesis of Indoleglycerol Phosphate Catalyzed by the α Subunit of Tryptophan Synthase from Escherichia coli

For the alpha subunit of tryptophan synthase and at constant concentration of D-glyceraldehyde 3-phosphate the saturation curves with respect to indole concentration are weakly sigmoidal. This phenomenon can be explained by interaction between indole bound to the effector site established previously and the active center of the monomeric alpha subunit. Kinetic studies of the inhibition of indoleglycerol phosphate synthesis by the analogue indolepropanol phosphate show that the inhibition is competitive with respect to D-glyceraldehyde 3-phosphate and non-competitive with respect to indole. Mechanisms with random addition of substrates or ordered addition with indole binding first can therefore be excluded. A quantitative fit of the data has been obtained to an ordered addition mechanism with D-glyceraldehyde 3-phosphate binding first and with a distribution of the enzyme between two states differing in V, governed by the binding of indole to the effector site. The kinetic constants obtained for the alpha subunit have been compared with those of the alpha 2 beta 2 complex of tryptophan synthase. Protein-protein interaction of the alpha subunit with the beta 2 subunit (a) does not alter the catalytic of the indoleglycerol phosphate synthesis, (b) suppresses the substrate activation by indole, and (c) changes the various equilibrium, rate and steady-state constants in the sense of conveying higher substrate specificity and catalytic efficiency to the alpha-subunit. The occurrence of local and gross conformational changes in the tryptophan synthase system is discussed.

The binding of indole to the alpha-subunit and beta2-subunit and to the alpha2beta2-complex of tryptophan synthase from Escherichia coli. Identification of a second indole-binding site on the alpha-subunit.

The binding of indole and indolepropanol phosphate, an analogue of the substrate indoleglycerol phosphate, to the individual alpha and beta2-subunits and to the alpha2beta2-complex of tryptophan synthase was studied by equilibrium dialysis. The use of [14C]indole and indolepropanol [32P]phosphate permitted simultaneous binding studies to be carried out. Competition between indole and indolepropanol phosphate in binding to a particular site was taken as evidence for that site being part of the active site of the alpha-subunit. The binding of indole to the active site of the alpha-subunit is weak (Kd = 18mM). A second distinct site binds indole more strongly (Kd = 1.5 mM) and interacts with the active site indirectly. It is therefore designated an effector site. Furthermore, the binding of indole and/or indolepropanol phosphate appears to stabilize different conformations of the alpha-subunit. The beta2-subunit binds indole only weakly (Kd = 12 mM) to many (n = 10) sites per polypeptide chain. The alpha2beta2-complex retains one or two sites per alphabeta-equivalent of relatively high affinity (Kd = 1.2 mM). The active sites of the component alpha and beta-subunits probably belong to the second class of many (n = 40) sites of low (Kd = 30 mM) affinity for indole. These findings support conclusions from the literature that both bi-substrate reactions involving indole catalyzed by tryptophan synthase and its subunits must follow strictly ordered addition mechanisms with the respective other substrate adding first.

Tryptophan Biosynthesis in Coprinus lagopus: A Genetic Analysis of Mutants

Thirty-one tryptophan-requiring mutants of Coprinus lagopus have been assigned by genetic and complementation analyses to four loci designated trp-I, trp-2, trp-3 and trp-4. The trp-1 and trp-3 loci were located in group III and trp-2 in group G of the linkage map. The trp-4 locus showed no linkage to the other trp loci or to markers in three additional linkage groups tested. From auxanographic tests and a study of accumulated biosynthetic intermediates, the enzymes controlled by each locus have been provisionally assigned. The trp-2 and trp-3 loci both appear necessary for anthranilate synthetase activity since mutants accumulated no intermediates. Only the trp-3 mutant could utilize anthranilic acid, therefore the trp-2 locus must also be involved in a subsequent step in the pathway. The trp-4 mutants utilized indole and accumulated anthranilic acid, and hence this locus is involved in the conversion of anthranilic acid to indoleglycerol phosphate. The trp-1 mutants utilized only tryptophan and accumulated indoleglycerol phosphophate and anthranilic acid. They are therefore blocked in the final steps of the pathway catalysed by tryptophan synthetase.

Some Physical Characteristics of the Enzymes of l-Tryptophan Biosynthesis in Higher Plants

Anthranilate synthetase, phosphoribosyltransferase, phosphoribosyl anthranilate isomerase, and indoleglycerol phosphate synthetase were examined in partially purified extracts of the monocotyledon, Zea mays and the dicotyledon, Pisum sativum. The plant extracts were chromatographed on DEAE-cellulose and Sephadex G150. The molecular weights of the enzymes were determined and found to be similar to those observed for many bacteria. None of the plant tryptophan enzyme activities was aggregated in vitro as is also the case with most bacteria. This is in contrast with the complex aggregation patterns observed in other eucaryotic organisms that have been examined (fungi and Euglena gracilis). The tryptophan enzymes from peas and corn were generally similar but some differences in stability were observed.

A rapid spectrophotofluorometric assay for indoleglycerol phosphate synthase

A rapid and sensitive spectrophotofluorometric assay for indoleglycerol phosphate synthase is presented. The method is based upon the fluorescence of the reaction product indoleglycerol phosphate and has two advantages over previously reported assays. It is more sensitive and is useful for measuring enzyme activities in extracts containing materials that prohibit the use of other methods.

The tryptophan synthase from Escherichia coli. An improved purification procedure for the alpha-subunit and binding studies with substrate analogues.

An improved method is described for the purification of the alpha-subunit of tryptophan synthase from Escherichia coli. The standard manganese chloride and acid-precipitation steps have been replaced by rapid and efficient chromatographic procedures. Indoleethanol phosphate, indoleprapanol phosphate and indolebutanol phosphate have been synthesized. They are not cleaved by tryptophan synthase and are strictly competitive inhibitors versus indoleglycerol phosphate. The inhibition constant decreases as the number of methylene groups in the side chain increases. This may reflect an improved accommodation of the indole and phosphate moienerated by binding indole, indoleglycerol phosphate and indolepropanol phosphate to the alpha-subunit are very similar. This reflects the transfer of the indole moiety to an hydrophobic environment within the active center. The binding of indolepropanol phosphate to the alpha2beta2-complex perturbs the spectrum of pyridoxal 5'-phosphate located in the beta2-subunit. This demonstrates direct or indirect interactions between the component active sites. Bind studies by spectrophotometric titration and equilibrium dialysis with indolepropanol [32P]phosphate show that there is only one binding site per equivalent of alpha-subunit. Complex formation with the beta2-subunit increases the affinity of the alpha-subunit for indolepropanol phosphate, It is a general consequence of protein-protein interaction in this system.

Subunit structure of anthranilate synthetase from Neurospora crassa.

Freshly purified preparations of anthranilate synthetase complex from Neurospora crassa appeared to be homogeneous on polyacrylamide disc gels and were composed of two distinct subunits, 94,000 and 70,000 daltons, respectively, as determined by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. Carboxymethylation of the complex or treatment with guanidine hydrochloride and urea before sodium dodecyl sulfate treatment did not alter the subunit pattern. When the purified complex was iodinated with 125I- or methylated with [14C]dimethylsulfate, no labeled components other than the two subunits stained with Coomassie blue were detected after electrophoresis in the presence of sodium dodecyl sulfate. Although some purified preparations were stable, most were unstable upon storage. Analysis of the unstable preparations on nondenaturing and sodium dodecyl sulfate polyacrylamide disc gels revealed that the complex in these preparations was progressively fragmented to smaller components and subunits upon repeated freeze-thaw treatment or prolonged incubation at or above 4 degrees. Distinct fragments were generated ranging in size down to 25,000 daltons, and some fragments retained some of the activities associated with the anthranilate synthetase complex. On the basis of these and earlier studies, we conclude that anthranilate synthetase from Neurospora crassa is composed of two distinct subunits in an alpha2beta2 structure; one subunit is a trifunctional peptide which contains the catalytic sites for the phosphoribosylanthranilate isomerase and indoleglycerol phosphate synthetase reactions, and associates with the second subunit to form glutamine-dependent anthranilate synthetase. The smaller subunits and components previously reported for this complex are apparently due to protease activity present in purified preparations.