Reaction of indole and analogues with amino acid complexes of Escherichia coli tryptophan indole-lyase: detection of a new reaction intermediate by rapid-scanning stopped-flow spectrophotometry

The effects of indole and analogues on the reaction of Escherichia coli tryptophan indole-lyase (tryptophanase) with amino acid substrates and quasisubstrates have been studied by rapid-scanning and single-wavelength stopped-flow spectrophotometry. Indole binds rapidly (within the dead time of the stopped-flow instrument) to both the external aldimine and quinonoid complexes with L-alanine, and the absorbance of the quinonoid intermediate decreases in a subsequent slow relaxation. Indoline binds preferentially to the external aldimine complex with L-alanine, while benzimidazole binds selectively to the quinonoid complex of L-alanine. Indole and indoline do not significantly affect the spectrum of the quinonoid intermediates formed in the reaction of the enzyme with S-alkyl-L-cysteines, but benzimidazole causes a rapid decrease in the quinonoid peak at 512 nm and the appearance of a new peak at 345 nm. Benzimidazole also causes a rapid decrease in the quinonoid peak at 505 nm formed in the reaction with L-tryptophan and the appearance of a new absorbance peak at 345 nm. Furthermore, addition of benzimidazole to solutions of enzyme, potassium pyruvate, and ammonium chloride results in the formation of a similar absorption peak at 340 nm. This complex reacts rapidly with indole to form a quinonoid intermediate very similar to that formed from L-tryptophan. This new intermediate is formed faster than catalytic turnover (kcat = 6.8 s-1) and may be an alpha-aminoacrylate intermediate bound as a gem-diamine.     

Study of coenzyme reorientation in the active center of tryptophanase by linear dichroism method

Tryptophanase from E.coli was oriented in a compressed slab of polyacrylamide gel and its linear dichroism (LD) and absorption spectra were measured. The free enzyme displays four LD bands at 305, 340, 425 and 490 nm. Two bands at 340 and 425 nm belong to the internal coenzyme-lysine aldimine. The 305 nm band apparently belongs to an aromatic amino acid residue; the sign and form of this band are changed upon the enzyme reaction with substrate analogs. The 490 nm band is present in the LD spectra of holo- and apoenzyme and disappears after treatment with NaBH4. It is suggested that the 490 nm band belongs to a quinoid enzyme subform. The reaction of tryptophanase with threo-beta-phenyl-DL-serine and L-threonine leads to formation of the external aldimine with a strong absorption band at 420-425 nm. The reduced LD (delta A/A) in this band is one order of magnitude greater than that in the 420 nm of the free enzyme. The complex with D-alanine is characterized by an intermediate LD value in the 425 nm band. In the presence of indole this complex displays the same LD as that observed with beta-phenylserine. The reaction of tryptophanase with L-alanine and oxindolyl-L-alanine leads to formation of the quinoid intermediate with a 500 nm absorption band. The LD value in this band differs from those in the absorption bands of the free enzyme. It is concluded that reorientations of the coenzyme occur in the course of the tryptophanase reaction.     

Linear dichroism studies of tryptophanase and its quasisubstrate complexes oriented in polyacrylamide gel

Tryptophanase from Escherichia coli was oriented in a compressed slab of polyacrylamide gel and its linear dichroism (LD) and absorption spectra have been measured. The free enzyme displays four LD bands at 305, 340, 425 and 490 nm. Two bands at 340 and 425 nm belong to the internal coenzyme-lysine aldimine. The 305-nm band apparently belongs to an aromatic amino acid residue. The 490-nm band disappears after treatment with NaBH4 or after incubation with L-alanine and subsequent dialysis. It is suggested that the 490-nm band belongs to a quinonoid enzyme subform. The reaction of tryptophanase with threo-3-phenyl-DL-serine, L-threonine and D-alanine leads to formation of an external aldimine with an intense absorption band at 420-425 nm. The values of reduced LD (delta A/A) in this band strongly differ from that in the 420-nm band of the free enzyme. The LD value of the complex with D-alanine is intermediate between those of the free enzyme and the complex with 3-phenylserine. In the presence of indole the complex with D-alanine displays the same LD as that observed with 3-phenylserine. The reaction of tryptophanase with L-alanine or oxindolyl-L-alanine leads to formation of a quinonoid intermediate with an absorption band near 500 nm. The LD value in this band is close to that of an external aldimine with L-threonine. It is concluded that reorientations of the coenzyme occur in the course of the tryptophanase reaction.     

Conformational changes in the active site of tryptophanase revealed by the circular dichroism method

Tryptophanase from E. coli displays positive CD in the coenzyme absorption bands at 337 and 420 nm. Breaking of the internal coenzyme-lysine imine bond upon reaction with hydroxylamine or amino-oxyacetate is accompanied by a strong diminution of the positive CD. Interaction of tryptophanase with L-threonine and beta-phenyl-DL-serine(threo form) leads to a decrease in absorbance at 337 nm and to an increase at 425 nm. This is associated with inversion of the CD sign, i.e. with disappearance of the positive CD in the 420-nm band and its replacement by a negative CD. L-Phenylalanine, alpha-methyl-DL-serine and D-alanine cause an increase in absorbance at 425-430 nm and a diminution of the positive CD in this band. In the presence of D-alanine and indole a negative CD appears in the 400-450 nm region. It is inferred that an external coenzyme-quasisubstrate aldimine is formed on interaction of the above amino acids with the enzyme. L-Alanine and oxindolyl-L-alanine evoke an intense narrow absorption band at 500 nm ascribed to a quinonoid intermediate; a positive CD is observed in this band. The dissymmetry factor delta A/A in the 500-nm band is much smaller than that in the absorption bands of the unliganded enzyme. Inversion of the CD sign on formation of the external aldimine and diminution of the dissymmetry factor in the quinonoid band indicate that reorientations of the coenzyme occur in the course of the catalytic action of tryptophanase.     

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.     

Inhibition of tyrosinase by indole compounds and reaction products. Protection by albumin.

Tyrosinase activity decreases as the reaction proceeds and is inhibited by L-3,4-dihydroxyphenylalanine oxidation products. Indole and tryptophan inhibit tyrosinase reaction and bovine albumin protects against end-product(s) inhibition or inactivation. Since the same tyrosinase reaction products are indole compounds and some authors reported the binding of indole derivatives with albumin, it is here suggested that indole intermediates of melanin synthesis inhibit or inactivate tyrosinase.     

A colorimetric method for the determination of indole, and its application to assay of tryptophanase.

Indole reacts with sodium nitrite and glycine-HCl buffer, pH 2.6, to form a red color that is stable for more than 1 week. The reaction is reproducible and is linear over a wide range of indole concentrations (0.05–1.00 μmol). Twelve indole derivatives, including tryptophan, and 17 protein amine acids do not interfere. Indole-3-acetic acid, indole-3-acrylic acid, indole-3-pyruvic acid, 5-indole carboxylic acid, and 5-hydroxyindole-3-acetic acid interfere to varying extents (16–27%). Free indole was determined in biological material containing tryptophan by the present method. The method is also applicable to the assay of tryptophanase activity without prior indole extraction.     

Physiological Effects of a Constitutive Tryptophanase in Bacillus alvei.

Hoch, J. A. (University of Illinois, Urbana), and R. D. DeMoss. Physiological effects of a constitutive tryptophanase in Bacillus alvei. J. Bacteriol. 90:604-610. 1965.-Tryptophanase synthesis in B. alvei is not under the control of tryptophan and is not subject to catabolite repression. Exogenously supplied tryptophan was converted to indole by tryptophanase, and was excreted into the culture medium. The amount of indole excreted was dependent upon the concentration of tryptophan supplied. At intermediate levels of tryptophan (5 to 15 mug/ml), the excreted indole was completely reutilized by the cell, in contrast to the result with higher levels. Indole reutil zation was shown to be dependent upon a functional tryptophan synthetase. In the absience of exogenous tryptophan, indole was excreted into the culture medium at an earlier physiological age. The early indole was shown not to be a consequence of tryptophanase action. The early indole accompanied uniformly the normal process of tryptophan biosynthesis, and the fission of indole-3-glycerol phosphate was suggested as the origin of the excreted indole.     

Indole can act as an extracellular signal to regulate biofilm formation of Escherichia coli and other indole-producing bacteria

We demonstrated previously that genetic inactivation of tryptophanase is responsible for a dramatic decrease in biofilm formation in the laboratory strain Escherichia coli S17-1. In the present study, we tested whether the biochemical inhibition of tryptophanase, with the competitive inhibitor oxindolyl-L-alanine, could affect polystyrene colonization by E. coli and other indole-producing bacteria. Oxindolyl-L-alanine inhibits, in a dose-dependent manner, indole production and biofilm formation by strain S17-1 grown in Luria-Bertani (LB) medium. Supplementation with indole at physiologically relevant concentrations restores biofilm formation by strain S17-1 in the presence of oxindolyl-L-alanine and by mutant strain E. coli 3714 (S17-1 tnaA::Tn5) in LB medium. Oxindolyl-L-alanine also inhibits the adherence of S17-1 cells to polystyrene for a 3-h incubation time, but mutant strain 3714 cells are unaffected. At 0.5 mg/mL, oxindolyl-L-alanine exhibits inhibitory activity against biofilm formation in LB medium and in synthetic urine for several clinical isolates of E. coli, Klebsiella oxytoca, Citrobacter koseri, Providencia stuartii, and Morganella morganii but has no affect on indole-negative Klebsiella pneumoniae strains. In conclusion, these data suggest that indole, produced by the action of tryptophanase, is involved in polystyrene colonization by several indole-producing bacterial species. Indole may act as a signalling molecule to regulate the expression of adhesion and biofilm-promoting factors.     

Alanine racemase of Pseudomonas. Observations on subtrate and inhibitor specificity

Systematic studies with purified alanine racemase and a number of substrate analogs permit the generalization that effective competitive inhibition is limited to 2- and 3-carbon compounds. A free α-amino group was not necessary for relatively tight binding; compounds lacking an amino group, or with an α-amino group acylated even by a bulky substituent, were bound as tightly as alanine. Substitution at the α-carbon of alanine (i.e., replacement of the α-H) eliminated binding, while substitution at the β-carbon generally reduced binding. Of several inhibitory compounds tested for substrate activity by H exchange with ³H₂O, only glycine appeared active. Covalent binding to the enzyme by halo analogs was not demonstrated.     

Catalytic studies on tryptophanase from Bacillus alvei

Tryptophanase from Bacillus alvei exhibited the expected spectrum of pyridoxal-5'-phosphate-dependent reactions. It exhibited l-serine dehydratase, S-alkyl-cysteine lyase, and cysteine desulfhydrase activities, as well as the classic tryptophanase reactions (all beta elimination reactions). It also acted as a tryptophan synthetase (beta replacement reactions) using indole plus l-serine or l-cysteine or S-methyl-cysteine as substrates. The beta elimination reactions are simple competitors of the replacement reactions for the same amino acid substrates. The kinetics of the reactions were examined in detail using a coupled continuous spectrophotometric assay. A product (indole) inhibition study of the beta elimination reaction with tryptophan showed simple, noncompetitive inhibition; the same study with allosubstrates showed noncompetitive inhibition by indole. These product studies provided data on the beta replacement reactions as well. The results are discussed in terms of a mechanism for the B. alvei tryptophanase.     

Interaction of tryptophanase with substrate analogs

Tryptophanase from Escherichia coli was studied with respect to its interactions with L-alanine, beta-chloro-L-alanine, L-phenylalanine, L-methionine, L-threonine, beta-phenyl-DL-serine (threo form) and also with a new tryptophan analog oxindolyl-L-alanine. Slow transamination of L-alanine in the active site of the enzyme was observed. Some evidence is presented which indicates that the side transamination reaction occurs during incubation of tryptophanase with an adequate substrate, beta-chloro-L-alanine. Absorption and circular dichroism (CD) spectra of the enzyme-quasisubstrate complexes have been recorded. Addition of beta-phenylserine and threonine to the enzyme induces a decrease of absorbance at 337 nm and an increase of absorbance at 420 nm. The spectral changes are associated with inversion of the CD sign, i.e. with disappearance of positive CD in the 420 nm band and appearance of negative CD in this band. It is inferred that beta-phenylserine and threonine form an external coenzyme-substrate aldimine which undergoes slow conversion to give a keto acid and the free enzyme. Addition of oxindolylalanine to tryptophanase results in the formation of an intense narrow absorption band at 504 nm with a shoulder at about 475 nm. This band belongs to a quinonoid intermediate. A positive CD is seen in the 504 nm band; the dissymmetry factor (delta A/A) in this band is much smaller than that in the absorption bands of the free enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Indole protects tryptophan indole-lyase, but not tryptophan synthase, from inactivation by trifluoroalanine.

Trifluoroalanine is a mechanism-based inactivator of Escherichia coli tryptophan indole-lyase (tryptophanase) and E. coli tryptophan synthase (R. B. Silverman and R. H. Abeles, 1976, Biochemistry 15, 4718-4723). We have found that indole is able to prevent inactivation of tryptophan indole-lyase by trifluoroalanine. The protection of tryptophan indole-lyase by indole exhibits saturation kinetics, with a KD of 0.03 mM, which is comparable to the KI for inhibition of pyruvate ion formation (0.01 mM) and the Km for L-tryptophan synthesis. Fluoride electrode measurements indicate the formation of 28 mol of fluoride ion per mole of enzyme during inactivation of tryptophan indole-lyase, and 121 mol of fluoride ion are formed per mole of enzyme in the presence of 2 mM indole during the same incubation period. 19F NMR spectra of reaction mixtures of tryptophan indole-lyase and trifluoroalanine showed evidence only for fluoride ion formation, in either the absence or the presence of indole, and difluoropyruvic acid was not detected. The partition ratio, kcat/kinact, is estimated to be 9. Tryptophan indole-lyase in the presence of trifluoroalanine exhibits visible absorption peaks at 446 and 478 nm, which decay at the same rate as inactivation. However, in the presence of 1 mM indole and trifluoralanine, tryptophan indole-lyase exhibits a peak only at 420 nm, and the spectra show a gradual increase at 300-310 nm with incubation. In contrast, tryptophan synthase is not protected by indole from inactivation by trifluoroalanine, and the absorption peak at 408 nm for the tryptophan synthase-trifluoroalanine complex is unaffected by indole. These results demonstrate that inactivation of tryptophan indole-lyase occurs via a catalytically competent species, probably the beta,beta-difluoro-alpha-aminoacrylate intermediate, which can be partitioned from inactivation to products by a reactive aromatic nucleophile, indole.     

Molecular orbital study on the interaction between coenzymes and enzymes; NAD+ or NADH and dehydrogenases

The absorption band at 260 mμ of NAD⁺ shifts to 360 mg by interaction with GAPDH or its analogues. Two explanations have been given on this red shift; one is an addition of such nucleophilic residue as sulfhydryl group in the enzyme to the position four in nicotinamide nucleus of NAD⁺, and the other is the charge transfer from such aromatic amino acid as tryptophan to NAD⁺. In the present paper, possibility of the charge transfer from indole residue to NAD⁺ was investigated quantum chemically. Taking into account of the electric field due to the charges in the enzyme, the absorption band of the NAD⁺-enzyme complex at 360 mμ was explained as a charge transfer from indole nucleus to NAD⁺. The blue shift of the absorption band of NADH at 340 mμ was also explained by taking into account of the electric field and this supported the proposition of Kosower (1962a).Stacking of adenine nucleus with indole nucleus in the NAD⁺-enzyme complex was suggested from the NMR spectroscopic data. Our molecular orbital calculations predicted that the effects of adenine on spectral shifts were not significant.     

Inhibition of tyrosinase by indole compounds and reaction products protection by albumin

Tyrosinase activity decreases as the reaction proceeds and is inhibited by L-3,4-dihydroxyphenylalanine oxidation products. Indole and tryptophan inhibit tyrosinase reaction and bovine albumin protects against end-products(s) inhibiton or inactivation. Since the same tyrosinase reaction products are indole compounds and some authors reported the binding of indole derivatives with albumin, it is here suggested that indole intermediates of melanin synthesis inhibit or inactivate tyrosinase.     

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.     

Sugar derivatives as new 6-phosphogluconate dehydrogenase inhibitors selective for the parasite Trypanosoma brucei

Sugar derivatives mimicking compounds which take part in the catalysed reaction have been assayed as alternative substrates and/or competitive inhibitors of 6-phosphogluconate dehydrogenase from Trypanosoma brucei and sheep liver. Phosphonate analogues have been synthesised and the new compound 5-deoxy-5-phosphono-D-arabinonate shows good selectivity towards the parasite enzyme. A number of 4-carbon and 5-carbon aldonates are strong inhibitors of the parasite enzyme with K(i) values below the substrate K(m) and some acyl derivatives are also potent inhibitors. At least five of the compounds showing a significant selectivity for the parasite enzyme represent leads for trypanocidal drugs against this recently validated target.     

Production L-tryptophan by Escherichia coli cells

Whole cells of Escherichia coli B 10 having high tryptophan synthetase activity were used directly as an enzyme source to produce L-tryptophan from indole and L- or D,L-serine. This strain is tryptophan auxotrophic, which is tryptophanase negative and, in addition, L- and D-serine deaminase negative under production conditions. To avoid inhibition of tryptophan synthetase by a high concentration of indole, nonaqueous organic solvents, Amberlite XAD-2 adsorbent, and nonionic detergents were used as reservoirs of indole in the reaction mixture for the production of L-tryptophan. As a result, different effects were observed on the production of L-tryptophan. Particularly, among the nonionic detergents, Triton X-100 was very efficient. Using Triton X-100 for production of L-tryptophan from indole and L- or D,L-serine by whole cells of Escherichia coli B 10, 14.14 g/100 mL and 14.2 g/100 mL of L-tryptophan were produced at 37 degrees C for 60 h.     

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.