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At least 19 recordsLinked to original sources
PubMed1976

Mutations in Escherichia coli that relieve catabolite repression of tryptophanase synthesis. Tryptophanase promoter-like mutations.

From a strain lacking adenyl cyclase and the catabolite-sensitive gene activator protein, two mutants were isolated that can synthesize tryptophanase. Each mutation is extremely closely linked to the tryptophanase structural gene. The mutations differ from one another in the rate of synthesis of tryptophanase that they permit in the genetic background in which they were isolated; they differ from one another and also from the wild type in the maximum rate of synthesis of tryptophanase that they permit in a genetic background with intact adenyl cyclase and catabolite-sensitive gene activator protein. Both mutations appear to lie in the tryptophanase promoter.

Chromosome Mapping↗
PubMed1986

Chemical modification of tryptophanase from E. coli with polyethylene glycol to reduce its immunoreactivity towards anti-tryptophanase antibodies.

Escherichia coli tryptophanase was modified with 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-s-triazine (activated PEG2, MW 5,000 x 2). The modified tryptophanase, in which approximately 43% of the total 120 amino groups and 38% of the total 16 sulfhydryl groups in the molecule were coupled, completely lost the immunoreactivity towards anti-tryptophanase serum from rabbit. Approximately 10% of the enzymic activity was retained. The modified enzyme showed the same physicochemical properties as the native enzyme: Km value for L-tryptophan (0.3 mmol/l), optimum pH (8.0) and optimum temperature (50 degrees C). The modified enzyme was more resistant than the native counterpart against proteolytic digestion with trypsin.

Animals↗
PubMed1976

Mutations in Escherichia coli that relieve catabolite repression of tryptophanase synthesis. Mutations distant from the tryptophanase gene.

Two mutants are described in which the synthesis of tryptophanase is unusually insensitive to catabolite repression. Neither mutation is linked by transduction to the tryptophane structural gene, neither mutation renders the synthesis of beta-galactosidase insensitive to catabolite repression, and the mutations do not permit tryptophanase to be synthesized in strains deficient in adenyl cyclase. During growth in glucose-minimal medium the mutants maintained a similar intracellular concentration of cyclic AMP to their wild-type parent; but since in the wild type the concentration of cyclic AMP was the same in glycerol-minimal medium as in glucose-minimal medium, it is doubtful whether catabolite repression is mediated by measurable changes in the concentration of this nucleotide.

Adenylyl Cyclases↗
PubMed1971

Cell-free synthesis of tryptophanase from Escherichia coli. Use of ribonucleic acid isolated from induced cells and a comparison of the product from a system employing ribosomes with that from one employing ribosomes and exogenous ribonucleic acid.

1. Polyribosomes and RNA were isolated from cultures in which tryptophanase (EC 4.2.1.-) was induced. The polyribosomes were incubated under conditions of protein synthesis, in the presence of a radioactive amino acid and a post-ribosomal supernatant fraction obtained from repressed cells. The RNA preparations were incubated under conditions of protein synthesis in the presence of a radioactive amino acid and a supernatant fraction containing ribosomes from repressed cells. 2. The system was characterized and the synthesis of a radioactive protein with the same chromatographic properties as tryptophanase was demonstrated. This synthesis was shown to be time-dependent and required the presence of RNA from induced cultures, ribosomes and an energy supply; it was inhibited by chloramphenicol. 3. The maximum activity for the synthesis of this protein was found to be associated with 23S rRNA isolated from sucrose gradients. 4. The N-terminal amino acid of tryptophanase was labelled in the protein synthesized in this system but not in the protein synthesized by polyribosomes (without added RNA). Conversely, the C-terminal amino acid of tryptophanase was labelled in the polyribosome system but not in the RNA-containing system. 5. Tryptic digests of protein labelled in vitro were compared with those of tryptophanase. No labelled tryptic peptides were identified other than tryptophanase tryptic peptides. An analysis of the results implied that in the polyribosome system almost the complete tryptophanase subunit chain was labelled but that in the RNA-containing system these chains were incompletely synthesized. 6. Sucrose-gradient analysis of protein synthesized in the RNA-containing system suggested that it cannot be converted into structures with the same sedimentation properties as native tryptophanase. 7. The significance of these results for the assay of tryptophanase mRNA and for an understanding of the control of the translation of this mRNA in vivo is discussed.

Amino Acids↗
PubMed1992

Effects of the chaperonin GroE on the refolding of tryptophanase from Escherichia coli. Refolding is enhanced in the presence of ADP.

The refolding of the tetrameric enzyme tryptophanase was facilitated by the chaperonin GroE. Maximum refolding yield of tryptophanase molecules (about 80%) was attained in the presence of a 15-fold excess of GroE 21-mer over tryptophanase monomer. The GroEL subunit was required for this improvement in refolding yield, whereas the GroES subunit was not. Light scattering experiments of the refolding reaction revealed that GroE bound to tryptophanase folding intermediates and suppressed their aggregation. The presence of ATP was required for the efficient dissociation of tryptophanase from GroEL. However, our experiments indicated that tryptophanase dissociated readily from GroEL in the presence of not only ATP, but also in the presence of non-hydrolyzable ATP analogues such as ATP gamma S (adenosine 5'-O-(3-thiotriphosphate)) and AMP-PNP (adenyl-5'-yl imidodiphosphate) as well. Surprisingly, the release of tryptophanase from GroEL was facilitated in the presence of ADP as well. We concluded that the binding of nucleotides such as ATP and ADP changed the conformation of GroEL and facilitated the dissociation of tryptophanase molecules. The conformation formed in the presence of ADP was distinct from the conformation formed in the presence of ATP, as shown by the selective dissociation of various folding proteins from the two conformations.

Adenine Nucleotides↗
PubMed1971

Catabolite repression of tryptophanase in Escherichia coli.

Catabolite repression of tryptophanase was studied in detail under various conditions in several strains of Escherichia coli and was compared with catabolite repression of beta-glactosidase. Induction of tryptophanase and beta-galactosidase in cultures grown with various carbon sources including succinate, glycerol, pyruvate, glucose, gluconate, and arabinose is affected differently by the various carbon sources. The extent of induction does not seem to be related to the growth rate of the culture permitted by the carbon source during the course of the experiment. In cultures grown with glycerol as carbon source, preinduced for beta-galactosidase or tryptophanase and made permeable by ethylenediaminetetraacetic acid (EDTA) treatment, catabolite repression of tryptophanase was not affected markedly by the addition of cAMP (3',5'-cyclic adenosine monophosphate). Catabolite repression by glucose was only partially relieved by the addition of cAMP. In contrast, under the same conditions, cAMP completely relieved catabolite repression of beta-galactosidase by either pyruvate or glucose. Under conditions of limited oxygen, induction of tryptophanase is sensitive to catabolite repression; under the same conditions, beta-galactosidase induction is not sensitive to catabolite repression. Induction of tryptophanase in cells grown with succinate as carbon source is sensitive to catabolite repression by glycerol and pyruvate as well as by glucose. Studies with a glycerol kinaseless mutant indicate that glycerol must be metabolized before it can cause catabolite repression. The EDTA treatment used to make the cells permeable to cAMP was found to affect subsequent growth and induction of either beta-galactosidase or tryptophanase much more adversely in E. coli strain BB than in E. coli strain K-12. Inducation of tryptophanase was reduced by the EDTA treatment significantly more than induction of beta-galactosidase in both strains. Addition of 2.5 x 10(-3)m cAMP appeared partially to reverse the inhibitory effect of the EDTA treatment on enzyme induction but did not restore normal growth.

Arabinose↗
PubMed1984

Tryptophanase synthesis in Escherichia coli: the role of indole replacement in supplying tryptophan and the nature of the constitutive mutation tnaR3.

The properties of the tryptophanase constitutive mutation tnaR3 have been investigated. It has previously been reported that mutants carrying tnaR3 grow poorly on medium that selects for constitutive expression of tryptophanase. Our results now show that this poor growth can be explained by the inability of tryptophanase to catalyse the synthesis of L-tryptophan from indole, pyruvate and ammonia at a rate sufficient to allow normal growth. Improved media for the characterization of tryptophanase constitutive mutants are described. The mutation tnaR3 rendered tryptophanase synthesis constitutive (at a different rate that at 37 degrees C is 30% of the fully induced wild-type) and not further inducible. Diploid studies showed that tnaR3 is cis-dominant, but no evidence was found for any effect in trans. In addition to rendering tryptophanase synthesis constitutive, tnaR3 affects the response of tryptophanase synthesis to different growth temperatures.

Escherichia coli↗
PubMed1991

Physiological studies of tryptophan transport and tryptophanase operon induction in Escherichia coli.

Escherichia coli forms three permeases that can transport the amino acid tryptophan: Mtr, AroP, and TnaB. The structural genes for these permeases reside in separate operons that are subject to different mechanisms of regulation. We have exploited the fact that the tryptophanase (tna) operon is induced by tryptophan to infer how tryptophan transport is influenced by the growth medium and by mutations that inactivate each of the permease proteins. In an acid-hydrolyzed casein medium, high levels of tryptophan are ordinarily required to obtain maximum tna operon induction. High levels are necessary because much of the added tryptophan is degraded by tryptophanase. An alternate inducer that is poorly cleaved by tryptophanase, 1-methyltryptophan, induces efficiently at low concentrations in both tna+ strains and tna mutants. In an acid-hydrolyzed casein medium, the TnaB permease is most critical for tryptophan uptake; i.e., only mutations in tnaB reduce tryptophanase induction. However, when 1-methyltryptophan replaces tryptophan as the inducer in this medium, mutations in both mtr and tnaB are required to prevent maximum induction. In this medium, AroP does not contribute to tryptophan uptake. However, in a medium lacking phenylalanine and tyrosine the AroP permease is active in tryptophan transport; under these conditions it is necessary to inactivate the three permeases to eliminate tna operon induction. The Mtr permease is principally responsible for transporting indole, the degradation product of tryptophan produced by tryptophanase action. The TnaB permease is essential for growth on tryptophan as the sole carbon source. When cells with high levels of tryptophanase are transferred to tryptophan-free growth medium, the expression of the tryptophan (trp) operon is elevated. This observation suggests that the tryptophanase present in these cells degrades some of the synthesized tryptophan, thereby creating a mild tryptophan deficiency. Our studies assign roles to the three permeases in tryptophan transport under different physiological conditions.

Amino Acid Transport Systems↗
PubMed1971

Biosynthesis in vitro of tryptophanase by polyribosomes from induced cultures of Escherichia coli.

1. Polyribosomes were isolated from Escherichia coli grown in media in which tryptophanase is induced and in which it is repressed. The polyribosomes from the induced bacteria had a small amount of tryptophanase activity associated with them. 2. A portion of the enzyme activity remained bound to polyribosomes during centrifuging in sucrose gradients. 3. Incubation of tryptophanase-containing polyribosomes with puromycin released enzyme activity. 4. The binding of the enzyme to the polyribosomes did not depend on the presence of DNA. 5. When the polyribosomes were incubated under conditions of protein synthesis with supernatant fraction obtained from repressed bacteria, a small but statistically significant increase in enzyme activity was produced. 6. When a radioactive amino acid was included in the incubation mixture for the tryptophanase system a radioactive protein was obtained whose chromatographic, electrophoretic and sedimentation properties were identical with those of tryptophanase. 7. The amount of incorporation was consistent with the amount of new enzyme synthesis predicted by the increase in enzyme activity. Both radioactive incorporation and increase in enzyme activity were shown to be energy-dependent and also negative controls were obtained by using zero-time incubations or polyribosomes isolated from either repressed cells or a mutant lacking the ability to produce tryptophanase. 8. The distribution of radioactive leucine in the carboxyl region of the newly labelled tryptophanase was examined by digesting the labelled protein with carboxypeptidases. It was shown that the radioactivity was more highly concentrated towards the carboxyl terminus when the incubation times for protein synthesis were shorter (implying that, with longer incubation times, longer lengths of polypeptide chain contained radioactive amino acid residues).

Bacterial Proteins↗
PubMed1975

Comparative studies on the properties of tryptophanase and tyrosine phenol-lyase immobilized directly on Sepharose or by use of Sepharose-bound pyridoxal 5'-phosphate.

Tryptophanase from Escherichia coli B/qt 7-A and tyrosine phenol-lyase (beta-tyrosinase) from Escherichia intermedia were immobilized on Sepharose 4B by several direct coupling reactions or through pyridoxal 5'-phosphate previously bound to Sepharose. The most active preparation of immobilized tryptophanase was obtained by coupling tetrameric apoenzyme to pyridoxal-P bound on Sepharose at the 6-position through a diazo linkage. This immobilization procedure involves the formation to Schiff base linkage between 4-formyl group of Sepharose-bound pyridoxal-P and the epsilon-amino group of the lysine residue at the active center of one subunit of tetrameric apo-tryptophanase, followed by the fixation of the Schiff base linkage by reduction with NaBH4. In the case of beta-tyrosinase having two catalytic centers, however, this method was not so suitable as the case of tryptophanase. Direct coupling of the apoenzyme to CNBr-activated Sepharose or to a bromoacetyl derivative of Sepharose gave better results. In each case, the affinity for substrate or coenzyme was scarcely influenced by the immobilization. When used repeatedly in a batch system or continuously in a flow system in the absence of added pyridoxal-P, immobilized holo-tryptophanase of holo-beta-tyrosinase gradually lost its original activity; however, supplement of pyridoxal-P to the reaction system restored its initial activity. From the kinetic analyses of these phenomena, the rate constants of coenzyme dissociation from immobilized tryptophanase and beta-tyrosinase were calculated. Upon immobilization, the pH optima of both enzymes shifted 0.5 to 1.0 pH unit to the alkaline side. Both immobilized enzymes showed higher thermal stability and resistance to a denaturing agent such as guinidine-HCl than their free counterpart. Furthermore, the reactivity of sulfhydryl group of beta-tyrosinase, in connection with its coenzyme-binding property, was conveniently studied by use of the immobilized enzyme.

Apoenzymes↗
PubMed1970

Physiological comparison of L-serine dehydratase and tryptophanase from Bacillus alvei.

Tryptophanase from Bacillus alvei also possesses serine dehydratase activity. A comparison of this enzyme with l-serine dehydratase [l-serine hydro-lyase (deaminating), EC 4.2.1.13] in toluene-treated whole cell preparations of the organism was undertaken. Tryptophanase is a constitutive enzyme in B. alvei. The dehydratase undergoes a repression-derepression-repression sequence as the l-serine level in the growth medium is increased from 0 to 0.1 m. Tryptophanase activity is decreased in organisms grown in medium containing glucose. Both enzymes are repressed in organisms grown in glycerol-containing medium. l-Serine dehydratase has a pH optimum of 7.5 in potassium phosphate buffer; tryptophanase functions optimally in this buffer at pH 8.2. Both enzymes lose activity in the presence of tris(hydroxymethyl)aminomethane buffer. Either K(+) or NH(4) (+) is required for full tryptophanase activity, but Na(+) is markedly inhibitory. These three cations are stimulatory to l-serine dehydratase activity. Both enzymes are subject to apparent substrate inhibition at high concentrations of their respective amino acids, but the inhibition of tryptophanase activity can be completely overcome by the removal of indole as it is formed. The dehydratase does not catalyze cleavage of d-serine, l-threonine, or alpha-substituted serine analogues at the concentrations tested. However, activity of the enzyme in cleaving l-serine is competitively inhibited by d-serine, indicating that the d-isomer can occupy an active site on the enzyme. The enzyme catalyzes cleavage of some beta-substituted serine analogues.

Ammonia↗
PubMed1970

Inhibition of the induced formation of tryptophanase in Escherichia coli by near-ultraviolet radiation.

Induced formation of tryptophanase in Escherichia coli B/r is temporarily inhibited by near-ultraviolet (UV) irradiation. The inhibition is greater when irradiation is at 5 C than when at room temperature. Hence, the inhibition is the result of a photochemical, rather than photoenzymatic, alteration of some cellular component. The action spectrum has a peak in the region of 334 nm and is similar to that for growth delay. However, inhibition of tryptophanase formation is more sensitive to near-UV irradiation than are growth, respiration, and the induced formation of beta-galactosidase. Thus, for tryptophanase the lack of formation cannot be due to general inhibition of metabolism. Pyridoxal phosphate absorbs in the near-UV region of the spectrum and is a cofactor for tryptophanase, but this enzyme in induced cells is not inactivated by near UV-radiations. An experiment in which toluene-treated suspensions from irradiated and unirradiated cells were mixed showed that irradiation does not cause the formation of an inhibitor of tryptophanase activity. The possibility remains that the absorption of radiant energy by pyridoxal phosphate interferes with the synthesis of tryptophanase.

Cold Temperature↗
PubMed1979

Complementation analysis of eleven tryptophanase mutations in Escherichia coli.

Nine independent mutants deficient in tryptophanase activity were isolated. Each mutation was transferred to a specialized transducing phage that carries the tryptophanase region of the Escherichia coli chromosome. The nine phages thus produced, and a tenth carrying a previously characterized tryptophanase mutation, were used to lysogenize a bacterial strain harbouring a mutation in the tryptophanase structural gene and also a suppressor of polarity. In no case was complementation observed; we conclude that there is no closely linked positive regulatory gene for tryptophanase.

Bacteriophage lambda↗
PubMed1978

Synthesis of tryptophanase in Escherichia coli: isolation and characterization of a structural-gene mutant and two regulatory mutants.

A mutant of E. coli has been isolated that is temperature-sensitive in respect of tryptophanase. When incubated at 60 degrees C, cell-free extracts of the mutant suffer inactivation of enzyme activity much more rapidly than similar extracts of the wild type. After lysogeny with a specialized transducing phage carrying the wild-type tryptophanase gene, the mutant is able to synthesize tryptophanase that is wild-type in its response to treatment at 60 degrees C. It is concluded that the mutation lies in the structural gene for the enzyme. Two further mutants have been isolated that synthesize tryptophanase constitutively. One mutation renders synthesis of the enzyme indifferent to the presence of inducer; the other mutation allows synthesis of the enzyme in the absence of inducer at about 35% of the fully induced wild-type rate. Neither mutation alleviates catabolite repression. Genetic mapping shows that the constitutive mutations lie very close to the structural-gene mutation, on the side of the structural gene distant from bglR.

Coliphages↗
PubMed1987

Reactions of O-acyl-L-serines with tryptophanase, tyrosine phenol-lyase, and tryptophan synthase.

The reactions of tryptophanase, tyrosine phenol-lyase, and tryptophan synthase with a new class of substrates, the O-acyl-L-serines, have been examined. A method for preparation of O-benzoyl-L-serine in high yield from tert.-butyloxycarbonyl (tBoc)-L-serine has been developed. Reaction of the cesium salt of tBoc-L-serine with benzyl bromide in dimethylformamide gives tBoc-L-serine benzyl ester in excellent yield. Acylation with benzoyl chloride and triethylamine in acetonitrile followed by hydrogenolysis with 10% palladium on carbon in trifluoroacetic acid gives O-benzoyl-L-serine, isolated as the hydrochloride salt. O-Benzoyl-L-serine is a good substrate for beta-elimination or beta-substitution reactions catalyzed by both tryptophanase and tyrosine phenol-lyase, with Vmax values 5- to 6-fold those of the physiological substrates and comparable to that of S-(o-nitrophenyl)-L-cysteine. Unexpectedly, O-acetyl-L-serine is a very poor substrate for these enzymes, with Vmax values about 5% of those of the physiological substrates. Both O-acyl-L-serines are poor substrates for tryptophan synthase, measured either by the synthesis of 5-fluoro-L-tryptophan from 5-fluoroindole and L-serine catalyzed by the intact alpha 2 beta 2 subunit or by the beta-elimination reaction catalyzed by the isolated beta 2 subunit. With all three enzymes, the elimination of benzoate appears to be irreversible. These results suggest that the binding energy from the aromatic ring of O-benzoyl-L-serine is used to lower the transition-state barrier for the elimination reactions catalyzed by tryptophanase and tyrosine phenol-lyase. Our findings support the suggestion (M. N. Kazarinoff and E. E. Snell (1980) J. Biol. Chem. 255, 6228-6233) that tryptophanase undergoes a conformational change during catalysis and suggest that tyrosine phenol-lyase also may undergo a conformational change during catalysis.

Catalysis↗
PubMed1975

Tryptophanase from Sphaerophorus funduliformis. Purification, molecular weight and subunit properties.

A crystalline tryptophanase can be obtained from extracts of Spaerophorus funduliformis using a heat treatment, hydroxyapatite chromatography and solubility in solutions of (NH4)2SO4 as a function of pH and temperature. The purified enzyme is homogeneous by several criteria. S. funduliformis tryptophanase has a specific activity of 11.5-13.5 and requires pyridoxal 5'-phosphate for enzymatic activity. Like other tryptophanases that have been studied, the S. funduliformis enzyme is a tetramer protein consisting of four apparently identical subunits. The native enzyme has a sedimentation coefficient of 11.2 S and a molecular weight of 244 000. In solutions of 5 M guanidine - HCl, 8 M urea, and sodium dodecylsulfate, at high pH or in the presence of thiols, the enzyme dissociates to 59 000 molecular weight species which are homogeneous by the criterion of weight. Peptide maps of the reduced holo-tryptophanase show one pyridoxal-containing peptide and, lacking agreement with the determined amino acid composition, suggest that the subunits of the enzyme contain a high degree of internal sequence homology.

Amino Acids↗