5-phosphoribosylpyrophosphate Phosphoribosyltransferase Research Articles

Evolution of aromatic amino acid biosynthesis and application to the fine-tuned phylogenetic positioning of enteric bacteria

A comprehensive phylogenetic tree for virtually the entire assemblage of enteric bacteria is presented. Character states of aromatic amino acid biosynthesis are used as criteria, and the results are compared with partial trees based upon sequencing of 16S rRNA, 5S rRNA, and tryptophan leader peptide. Three major clusters are apparent. Enterocluster 1 possesses a gene fusion (trpG-trpD) encoding anthranilate synthase: anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase of tryptophan biosynthesis. This cluster includes the genera Escherichia, Shigella, Citrobacter, Salmonella, Klebsiella, and Enterobacter. The remaining two clusters lack the trpG-trpD gene fusion, but differ in the presence (enterocluster 2) or absence (enterocluster 3) of the three-step overflow pathway to L-phenylalanine. Enterocluster 2 consists of the genera Serratia and Erwinia. Enterocluster 3 includes the genera Cedecea, Kluyvera, Edwardsiella, Hafnia, Yersinia, Proteus, Providencia, and Morganella. Within these three major clusters, a tentative hierarchy of subcluster ordering is formulated on the basis of all data available. This hierarchical framework is proposed as a general working basis for continued refinement of the phylogenetic relationships of enteric bacteria.

Utility of a Bifunctional Tryptophan Pathway Enzyme for the Classification of the Herbicola-Agglomerans Complex of Bacteria

Bifunctional proteins are the result of relatively infrequent genetic events that are faithfully conserved. They are reliable markers that define phylogenetic clusters. The bifunctional protein anthranilate synthase: anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase (AS:PRT) separates two enteric clusters. Previously published data show that this bifunctional protein is present in the lineage shared by Escherichia coli, Salmonella typhimurium, Citrobacter freundii, Klebsiella pneumoniae, Enterobacter aerogenes, and Enterobacter cloacae, but is absent in Erwinia carotovora, Serratia marcescens, Proteus vulgaris, Morganella morganii, and Hafnia alvei. It has been postulated that aerogenic and anaerogenic strains of Enterobacter agglomerons belong to different groups, which correspond to the clusters mentioned above, respectively. In confirmation of this, we found that Enterobacter agglomerons ATCC 29915 (aerogenic) possesses bifunctional AS:PRT, whereas Enterobacter agglomerons ATCC 27155T (T = type strain) (anaerogenic) lacks AS:PRT. We also found that Erwinia herbicola 33243T lacks AS:PRT. Beji et al. (Int. J. Syst. Bacteriol. 38:77-88) recently showed that the type strains of Enterobacter agglomerons (ATCC 27155) and Erwinia herbicola (ATCC 33243) belong to the same genomic species. We suggest that Enterobacter agglomerons (= Erwinia herbicola) should be excluded from the genus Enterobacter. We further suggest that strains currently designated Enterobacter agglomerons can be grouped with Erwinia herbicola or with the enteric cluster containing Enterobacter depending upon whether bifunctional AS:PRT is absent or present, respectively.

A mutation to 5-methyltryptophan dependence in the trp operon of Salmonella typhimurium

The AS-PRT enzyme complex which catalyzes the first two steps in the biosynthesis of tryptophan in S. typhimurium consists of two polypetide subunits: anthranilate synthetase (component I or AS-CoI) and anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase (PRT). These polypeptides are the products of the first two structural genes of the trp operon, trpA and trpB respectively. The PRT component has two functions: the aminoterminal 40% of the polypeptide is necessary for glutamine amidotransferase activity (GAT) while the carboxy-terminal 60% carries out the PRT activity proper. The mutant strain SO495 has a mutation, trpA515, which confers a unique phenotype: while the strain is capable of utilizing anthranilic acid (AA) a substrate of PRT, as a growth factor, it can only do so in the presence of the analogue 5-methyltryptophan (MT) normally a potent growth inhibitor. Previous evidence indicates that SO495 may possess a somewhat altered PRT, and that its activity could be inhibited by an altered, enzymatically inactive AS made in this strain under derepression. Some experiments designed to test these possibilities are described in this paper. Various properties of the PRT's of the MT-dependent mutant and several of its MT-independent revertants were examined and compared. These included the determination of their apparent Km's for the substrates anthranilic acid (AA) and phosphoribosyl pyrophosphate (PRPP) and the presence or absence of GAT activity. In addition, the possibility that a complex consisting of PRT and an enzymatically inactive AS-CoI was present in some of the revertant strains only when grown under derepressing conditions was investigated by gel chromatography. The results showed that the MT-dependent strain SO495 and the MT-independent revertants have PRT's which differ from each other as well as from wild type LT7 PRT. In MT-independent revertants which retain the trpPO region and most of trpA, PRT can form a loose aggregate which elutes from Bio-Gel columns as three fast moving peaks. This loose aggregate is absent when the strains are grown under repressing conditions and is always absent in strains which lack most of the trpA gene. These results support the idea that the dependence of strain S0495 on MT for utilization of AA as a growth factor has to do with the inhibition of the altered PRT made in this mutant by an altered AS-CoI polypeptide which is synthesized only under derepression. They also suggest that translation of trpB starts from different points in the wild type, S0495 and the MT-independent revertants.

Immunochemical analysis of the anthranilate synthase-anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase aggregate of Escherichia coli

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTImmunochemical analysis of the anthranilate synthase-anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase aggregate of Escherichia coliMichael Largen and Stanley E. MillsCite this: Biochemistry 1977, 16, 11, 2526–2532Publication Date (Print):May 31, 1977Publication History Published online1 May 2002Published inissue 31 May 1977https://doi.org/10.1021/bi00630a032RIGHTS & PERMISSIONSArticle Views15Altmetric-Citations3LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (901 KB) Get e-Alerts Get e-Alerts

Anthranilate synthase-anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase from Salmonella typhimurium: Purification of the enzyme complex and analysis of multiple forms

1. 1.|The anthranilate synthase-anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase enzyme complex (chorismate pyruvate-lyase (aminoaccepting), EC 4.1.3.27) · ( N-(5′- phosphoribosyl)-anthranilate :pyrophosphate phosphoribosyltransferase, EC 2.4.2.18), from Salmonella typhimurium has been purified with high yields to homogeneity. Sodium dodecyl sulfate gel electrophoresis of the purified enzyme complex revealed one major band containing 96% of the protein. The final yield of enzyme complex activity ranged from 30 to 60%. The absorbance spectrum of enzyme complex showed a peak at 280 nm and fine structure with peaks at 253, 259, 266 and 269 nm. These latter wavelengths correspond closely with the known absorbance maxima of phenylalanine. 2. 2.|When purified enzyme complex was subjected to standard gel electrophoresis, a four band pattern of protein peaks was consistently observed. The major enzyme complex band was apparently the native tetramer, having a molecular weight of 280000 and containing ammonia- and glutamine-dependent anthranilate synthase activity. The other three bands were molecular weight isomers of the major enzyme complex band. Two forms of molecular weight isomers were present: dimers and an aggregate of the native enzyme complex. The molecular weight isomers of the enzyme complex may represent forms generated by aggregation and denaturation of the native enzyme complex. 3. 3.|A new and highly sensitive spectrophotometric assay for phosphoribosyltransferase is described. The method is based upon the difference in extinction coefficients between anthranilate and N-(5′- phosphoribosyl)anthranilate .

Metal ion requirement and tryptophan inhibition of normal and variant anthranilate synthase-anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase complexes from Salmonella typhimurium

1. 1.|Both Mn 2+ and Co 2+ can replace Mg 2+ as the required divalent cation for all activities of the enzyme complex between anthranilate synthase (chorismate pyruvate-lyase (amino-accepting), EC 4.1.3.27) and anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferase ( N-(5′- phosphoribosyl)-anthranilate :pyrophosphate phosphoribosyltransferase, EC 2.4.2.18) from Salmonella typhimurium. They have much lower, apparent K m values than Mg 2+, both for glutamine-dependent anthranilate synthase ( Mn 2+ = 1.1 μM , Co 2+ = 2.6 μM , Mg 2+ = 83 μM ) and for phosphoribosyltransferase ( Mn 2+ = 16 μM , Co 2+ = 14.6 μM, Mg 2+ = 133 μM ). The ratio of total Mg 2+ to total Mn 2+ found in a cell extract of S. typhimurium trpE2, the source of normal enzyme complex, was found to be 350, suggesting that Mg 2+ is probably utilized by the enzyme complex in vivo under our growth conditions. 2. 2.|An enzyme complex has been isolated from a mutant strain of S. typhimurium (SO-515) that has a variation in the anthranilate synthase subunit which is thought to be a single amino acid substitution. This variation causes glutamine-dependent anthranilate synthase to be hypersensitive to feedback inhibition by tryptophan ( K i = 0.4 μM compared to K i = 20 μM for normal enzyme complex). The phosphoribosyltransferase in the variant enzyme complex is also hypersensitive to tryptophan but the kinetics are complex and involve activation by tryptophan in the presence of low amounts of 5-phosphoribosyl 1-pyrophosphate. 3. 3.|In the variant enzyme complex the apparent K m for Mg 2+ is elevated to 360 μM for glutamine-linked anthranilate synthase but reduced to 75 μM for phosphoribosyltransferase. 4. 4.|These results suggest that the variant enzyme complex has altered tertiary and quaternary structures and that regulation of both activities is effected by tryptophan binding to only anthranilate synthase.

Tryptophan biosynthesis in Salmonella typhimurium: location in trpB of a genetic difference between strains LT2 and LT7

Salmonella typhimurium prototrophs carrying a trpR mutation synthesize tryptophan biosynthetic enzymes constitutively. When feedback inhibition of anthranilate synthetase but not 5'-phosphoribosylpyrophosphate phosphoribosyltransferase activity was by-passed by growing cells on media supplemented with anthranilic acid, all trpR prototrophs overproduced and excreted tryptophan. However, the rate of tryptophan production depended on both the ancestry of the trpR strain and the integrity of its trpA gene. Prototrophs with trp genes derived from S. typhimurium strain LT2 produced tryptophan more efficiently than those with trp genes derived from strain LT7. This strain difference was cryptic insofar as it did not affect the growth rate; it was revealed only as a rate-limiting step in the constitutive biosynthesis of tryptophan in the presence of anthranilic acid, and was due to a lesion in the LT7-derived trpB gene. Strains with LT7-derived trp genes bearing a deletion in trpA produced tryptophan as readily as LT2 trpR prototrophs. This indicated that LT7-specific 5-phosphoribosylpyrophosphate phosphoribosyltransferase must be aggregated with the trpA gene produce to give an observable reduction of constitutive tryptophan production. The discovery of this strain difference has particular implications for studies involving the activities of trpA and B genes and their products in S. typhimurium and may have general significance for other studies involving different strains of Salmonella.

Anthranilate synthetase-anthranilate 5-phosphoribosylpyrophosphate phosphoribosyl-transferase from Salmonella typhimurium: Inactivation of glutamine-dependent anthranilate synthetase by agarose-bound anthranilate

Exposure of the anthranilate synthetase-anthranilate phosphoribosyltransferase enzyme complex (chorismate pyruvate-lyase (amino-accepting) EC 4.1.3.27 and N-(5′-phosphoribosyl)-anthranilate pyrophosphate phosphoribosyl-transferase, EC 2.4.2.18) from Salmonella typhimurium to agarose-bound anthranilate led to the slow inactivation of glutamine-dependent anthranilate synthetase activity, an activity dependent on protein-protein interaction in the enzyme complex. Region I of phosphoribosyltransferase, the location of the enzyme complex glutaminase activity, is the site of alteration. Phosphoribosyl-transferase and NH 3-dependent anthranilate synthetase activities and tryptophan regulation of phosphoribosyltransferase were unaffected by the anthranilate matrix. The molecular weight (280 000) and subunit molecular weight (62 000) of the enzyme complex eluted from an anthranilate matrix were identical to those of enzyme complex purified by classical methodology. The enzyme complex could be partially protected against inactivation by storing in 0.1 M l-glutamine or 30% glycerol and completely protected by storing in 50% glycerol at −18°C. Evidence is presented that the anthranilate matrix acts as a hydrophobic matrix and may be binding to and altering a hydrophobic region in the enzyme complex. The anthranilate matrix provides a convenient tool for altering a specific region of an enzyme complex without covalent modification. At the same time, the results demonstrate that affinity matrices are not necessarily innocuous but may subject macromolecules to an adverse environment not previously recognized.

Fluorescent assay of anthranilate synthetase-anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase enzyme-complex on polyacrylamide gels

A rapid and sensitive method for the localization of anthranilate synthetaseanthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase enzyme-complex from Salmonella typhimurium after disc gel electrophoresis has been developed, based on the fluorescence of the anthranilate synthetase product, anthranilate. Both NH 3-and glutamine-dependent anthranilate synthetase activities could be detected. A linear transport attachment on a Gilford Spectrophotometer was modified to scan the fluorescent gels. Semiquantitative evaluation of enzyme-complex activity was achieved by interpreting the chart recorder tracings of assayed gels. The enzyme-complex on polyacrylamide gels can be assayed under various conditions. The concentration of tryptophan required for half-maximal inhibition of NH 3-dependent anthranilate synthetase activity was determined from enzyme-complex assayed on polyacrylamide gels and in solution. A value of 4–8 μ m tryptophan was determined in both cases. The technique is applicable to any enzyme that forms a fluorescent product.

Monomeric and dimeric forms of component II of the anthranilate synthetase--anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase complex of Salmonella typhimurium. Implications concerning the mode of assembly of the complex.

Monomeric and dimeric forms of component II of the anthranilate synthetase--anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase complex of Salmonella typhimurium. Implications concerning the mode of assembly of the complex.

Use of affinity matrices in determining steric requirements for substrate binding: Binding of anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase from Salmonella typhimurium to sepharose-anthranilate derivatives

The binding of anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase (PRT) in crude extracts of Salmonella typhimurium to Sepharose-anthranilic acid derivatives has been studied. When anthranilic acid was covalently bound to hydrocarbon “arms” by its free amino group or phenyl moiety, the derivatives thus prepared bound PRT preferentially. PRT activity could be eluted from these adsorbents using buffers of increasing pH. The specific activity of PRT eluted from these Sepharose-anthranilate adsorbents was 10- to 12-fold that of the original extract. Anthranilic acid bound to aminohexamethylimino-Sepharose by its free carboxyl group did not bind PRT effectively. Results indicated that the carboxyl group of anthranilic acid must be free in order for PRT to bind the Sepharose-anthranilate derivatives. Throughout this study, control columns consisting of Sepharose derivatives containing the appropriate hydrocarbon “arms” alone were used to investigate the possibilities of nonspecific enzyme binding. The findings obtained stress the necessity of using such controls in any affinity studies of this type. The prospect of utilizing the techniques of affinity chromatography to examine the requirements for the binding of proteins to specific ligands is discussed.

Purification of anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase from [formula omitted] using affinity chromatography: Resolution of monomeric and dimeric forms

Anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase (PRT) as the unaggregated enzyme, has been purified to 90–95 percent homogeneity using affinity and ion-exchange chromatography. The affinity matrix consisted of anthranilate coupled to succinylamidohexamethylimino-Sepharose via its free amino group. Electrophoresis of the purified PRT on gels of varying polyacrylamide concentrations resolved a faster-moving minor band at concentrations above 8 percent. Both the major and minor bands were found to possess PRT activity. The minor band was found to have a molecular weight one-half that of the major band. Electrophoresis of the enzyme in the presence of sodium dodecyl sulfate resulted in almost complete conversion to the lower molecular weight form.

Anthranilate Synthetase from Serratia marcescens: ON THE PROPERTIES AND RELATIONSHIP TO THE ENZYME FROM SALMONELLA TYPHIMURIUM

Abstract Anthranilate synthetase was purified from Serratia marcescens. The enzyme was homogeneous by the criterion of disc gel electrophoresis. Glutamine- and NH3-dependent anthranilate synthetase activities were subject to end product inhibition by tryptophan. Inhibition by tryptophan exhibited positive cooperativity. In the absence of tryptophan, Michaelis-Menten kinetics were obtained for saturation by the two substrates, chorismate and glutamine. Positive cooperativity for chorismate was detected in the presence of tryptophan. These kinetic properties of anthranilate synthetase from S. marcescens were similar to those of the multifunctional anthranilate synthetase-anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase (PR transferase) aggregate from Salmonella typhimurium. A molecular weight of approximately 141,000 was estimated for the oligomeric anthranilate synthetase from S. marcescens. Nonidentical subunits of molecular weights approximately 60,000 and 21,000 were detected by disc gel electrophoresis in sodium dodecyl sulfate. The glutamine analogue, 6-diazo-5-oxo-l-norleucine (DON), and sulfhydryl reagents selectively inactivated glutamine-dependent anthranilate synthetase activity; NH3-dependent activity was largely retained. 14C-Labeled DON and iodo[14C]acetamide were incorporated into the small subunit thus establishing the relationship of this subunit to anthranilate synthetase Component II from anthranilate synthetase-PR transferase of S. typhimurium. Approximately 2 moles of DON or iodoacetamide were incorporated per mole of anthranilate synthetase, suggesting that the oligomeric enzyme contains two glutamine-binding subunits (anthranilate synthetase Component II). From a consideration of the approximate size of the native enzyme and its subunits a composition of two chains each of anthranilate synthetase Components I and II is suggested. A glutaminase activity was detected. This activity may serve to transfer the amide of glutamine from anthranilate synthetase Component II to the catalytic site on Component I. Catalytic properties and structural features of anthranilate synthetase from S. marcescens were compared to those of trypsin-treated anthranilate synthetase-PR transferase from S. typhimurium (Hwang, L. H., and Zalkin, H., J. Biol. Chem., 246, 2338 (1971)). On the basis of similarities between the two enzymes it is suggested that gene fusion could account for the two types of anthranilate synthetase.

On the Composition of Anthranilate Synthetase-Anthranilate 5-Phosphoribosylpyrophosphate Phosphoribosyltransferase from Salmonella typhimurium

Abstract The molecular weight of purified anthranilate synthetase-anthranilate 5-phosphoribosylpyrophosphate phosphoribosyl-transferase (PR transferase), determined by sedimentation equilibrium was 280,000. A molecular weight of approximately 62,000 was obtained for each of the nonidentical subunits, anthranilate synthetase Component I and PR transferase (anthranilate synthetase Component II) by sodium dodecyl sulfate gel electrophoresis. The two types of subunits were separated by disc gel electrophoresis in 8 m urea. Anthranilate synthetase-PR transferase contained approximately equal amounts of each subunit. Two high affinity tryptophan-binding sites which exhibited positive cooperativity were detected. Binding of tryptophan to these sites was antagonized by both chorismate and Mg2+. Two chorismate-binding sites were detected which had a dissociation constant equal to the Km value for chorismate in the anthranilate synthetase reaction. Binding of chorismate was strongly antagonized by tryptophan. These properties establish that binding of chorismate and tryptophan was to anthranilate synthetase Component I of the aggregate. It is proposed that end product inhibition of anthranilate synthetase activity by tryptophan is due to antagonism of chorismate binding. The results of binding studies and of subunit separation indicate that anthranilate synthetase-PR transferase is composed of two polypeptide chains each of anthranilate synthetase Components I and II.

Multiple Forms of Anthranilate Synthetase-Anthranilate 5-Phosphoribosylpyrophosphate Phosphoribosyl-transferase from Salmonella typhimurium

Abstract Extracts of Salmonella typhimurium prepared by different techniques contain both soluble and particulate forms of the anthranilate synthetase-anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferase (PR transferase) aggregate. The particulate form of the enzyme may be attached to fragments of the cell envelope. PR transferase activity of the particulate enzyme aggregate is partly masked. Other properties of the two forms are similar. The particulate enzyme was solubilized by repeated washing or by treatment with trypsin or crude lipase. Lipase- and trypsin-treated enzymes were purified to apparent homogeneity as judged by acrylamide gel electrophoresis and appeared to be similar. Both lacked PR transferase activity. The molecular weights of the lipase- and trypsintreated enzymes were approximately 141,000 compared with approximately 261,000 for the untreated enzyme aggregate. This decrease is accounted for by digestion of PR transferase chains of molecular weight approximately 62,000 to molecular weight of about 15,000 to 19,000. The digested PR transferase polypeptide chain of molecular weight approximately 15,000 to 19,000 contains a functional glutamine site and interacts with anthranilate synthetase Component I to give a functional glutamine-dependent anthranilate synthetase activity that is indistinguishable from that of the native enzyme.

The Anthranilate Synthetase-Anthranilate-5-Phosphoribosylpyrophosphate Phosphoribosyltransferase Aggregate: ON THE REACTION MECHANISM OF ANTHRANILATE SYNTHETASE FROM SALMONELLA TYPHIMURIUM

Abstract The reaction of the glutamine analogue, 6-diazo-5-oxo-l-norleucine (DON), with the bifunctional, regulatory enzyme aggregate of the tryptophan pathway, anthranilate synthetase-anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase (PR transferase), and with the anthranilate synthetase Component I protein of the aggregate was studied. DON inhibits anthranilate synthetase Component I activity and glutamine- and NH3-dependent anthranilate synthetase activities of anthranilate synthetase-PR transferase. DON inactivates glutamine-dependent anthranilate synthetase activity of the enzyme aggregate, but has less effect on the NH3-dependent anthranilate synthetase activity of the aggregate and does not inactivate anthranilate synthetase Component I. Inactivation of anthranilate synthetase activity of anthranilate synthetase-PR transferase by DON is dependent upon the substrate chorismate, thus providing evidence for ordered binding of first chorismate and then DON (or glutamine). Inactivation of anthranilate synthetase by DON is prevented by the feedback inhibitor, tryptophan. It is suggested that tryptophan evokes feedback inhibition, at least in part, by either preventing chorismate from binding or by preventing the conformational change needed to allow glutamine to bind. The two components of the aggregate, anthranilate synthetase Component I and PR transferase, can be separated by disc gel electrophoresis in 8 m urea. Following treatment of anthranilate synthetase-PR transferase with 14C-DON, most of the radioactivity is incorporated into PR transferase. It therefore appears that the two substrates for anthranilate synthetase bind to different polypeptide chains, chorismate to anthranilate synthetase Component I and glutamine to PR transferase. Approximately 2 moles of 14C-DON bind per mole of enzyme; this suggests a subunit composition of (anthranilate synthetase Component I)2 (PR transferase)2. Titrations with 5,5'-dithiobis(2-nitrobenzoic acid) indicate that DON alkylates 2 cysteine residues per enzyme molecule. A glutaminase activity has been detected in purified preparations of anthranilate synthetase-PR transferase. Glutaminase activity is stimulated by chorismate and inhibited by tryptophan. It is suggested that this activity serves to transfer the amide of glutamine from the site on PR transferase to the NH3 site on anthranilate synthetase Component I. A reaction mechanism which accommodates the experimental data is proposed.

The Anthranilate Synthetase-Anthranilate 5-Phosphoribosylpyrophosphate Phosphoribosyltransferase Aggregate: PURIFICATION OF THE AGGREGATE AND REGULATORY PROPERTIES OF ANTHRANILATE SYNTHETASE

Abstract Relatively stable, highly purified preparations of the anthranilate synthetase-anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase aggregate have been obtained from Salmonella tryphimurium. A molecular weight of approximately 285,000 was estimated for the enzyme aggregate by sucrose gradient centrifugation. Anthranilate synthetase activity was completely inhibited by 10 µm tryptophan under appropriate conditions. Inhibition by tryptophan was competitive with chorismate and noncompetitive with glutamine and showed positive cooperativity. Anthranilate synthetase activity was almost completely insensitive to inhibition by tryptophan in the presence of 5.0 mm Mg2+, suggesting distinct substrate and inhibitor sites. Mg2+ was required for enzymatic activity. Inhibition of enzyme activity was obtained by low concentrations of anthranilate, indicating possible regulatory significance. This inhibition was partially overcome by excess Mg2+, suggesting that inhibition was due, at least in part, to interaction of anthranilate with the tryptophan regulatory site. Positive cooperativity obtained for anthranilate was similar to that obtained for tryptophan. In the presence of anthranilate or tryptophan positive cooperativity for chorismate was observed. For the NH3-dependent anthranilate synthetase activity of the anthranilate synthetase-anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase aggregate, negative cooperativity for NH3 was indicated by the shapes of plots of initial velocity against (NH4)2SO4 concentration and Lineweaver-Burk plots and the values for related kinetic constants. Initial velocity experiments were consistent with a sequential mechanism.

The Anthranilate Synthetase-Anthranilate 5-Phosphoribosylpyrophosphate Phosphoribosyltransferase Aggregate: CATALYTIC AND REGULATORY PROPERTIES OF AGGREGATED AND UNAGGREGATED FORMS OF ANTHRANILATE 5-PHOSPHORIBOSYLPYROPHOSPHATE PHOSPHORIBOSYLTRANSFERASE

Abstract Anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase (PR transferase) catalyzes the second specific reaction in the tryptophan biosynthetic pathway. This protein is aggregated to anthranilate synthetase Component I, which functions in the catalysis of the preceding reaction in Salmonella tryphimurium. Catalytically active preparations of unaggregated PR transferase had a molecular weight of approximately 87,000. The denatured polypeptide chain had a molecular weight of approximately 63,000, suggesting that the unaggregated enzyme is a single polypeptide chain. Aggregated and unaggregated forms of PR transferase activity were inhibited by tryptophan and required saturating concentrations of substrates for maximal inhibition. Chorismate, a substrate for anthranilate synthetase Component I, antagonized the inhibition of aggregated PR transferase activity by tryptophan but not the tryptophan inhibition of the unaggregated form of the enzyme. Indications of negative cooperativity for anthranilate were observed for aggregated PR transferase in the presence of tryptophan. A partial requirement for Mg2+ was shown for the enzymatic activity and tryptophan inhibition of aggregated PR transferase but not for the unaggregated enzyme. The pH profiles for inhibition by tryptophan differed for the two forms of the enzyme although the pH optima for enzyme activity were similar. The Km values for 5-phosphoribosyl 1-pyrophosphate and anthranilate were 6.7 µm and 8.3 µm, respectively, for the aggregated enzyme and they deviated less than 2-fold for the unaggregated PR transferase. Initial velocity patterns for saturation by substrates of aggregated PR transferase activity appeared parallel but a ping-pong mechanism was excluded by the pattern of PPi product inhibition and lack of exchange of 32PPi into 5-phosphoribosyl 1-pyrophosphate. The patterns of product inhibition by PPi differed for the two forms of PR transferase. It was concluded from presently available evidence that the native anthranilate synthetase-PR transferase aggregate has the composition (anthranilate synthetase)2 (PR transferase)2 and that the PR transferase polypeptide chains, similar to the anthranilate synthetase polypeptide chains, contain sites for substrates and end product inhibitor.