Trypotophanase from a marine bacterium, Vibrio K-7 synthesis, purification and some chemical catalytic properties.

The conditions for synthesis, purification, and properties of tryptophanase by a marine organism (Vibrio K-7) were studied. Tryptophanase was induced by tryptophan and its analogs, and partially repressed by 0.5% glucose or glycerol. NaCl (0.4 M) was required for optimal growth and tryptophanase activity in whole cells. The enzyme was purified to 92% homogeneity by heat treatment, hydroxyapatite chromatography and fractionation with ammonium sulfate. This tryptophanase has been found to have kinetic properties similar to the tryptophanase from other microorganisms. It carries out both alpha, beta-elimination reactions (using tryptophan, serine, cysteine and S-methylcysteine as substrates) and beta-replacement reactions (forming tryptophan from indole and serine, cysteine or S-methyl-cysteine). The enzyme has a sedimentation coefficient of 9.2S and requires pyridoxal 5'-phosphate as a cofactor. The optimal pH for the tryptophanase reaction is pH 8.0.

Effect of microflora on the free amino acid distribution in various regions of the mouse gastrointestinal tract.

The distribution of free amino acids in the contents of various regions of the gastrointestinal tract (stomach, upper small intestine, lower small intestine, cecum, upper colon and lower colon) was studied in germfree and conventionalized mice. Particular emphasis was placed on the conversion of tryptophan to indole as a probe for studying intermicrobial interactions and microbe-host interactions in vivo. Great differences were observed in the free amino acid content of the various regions of the digestive tract in each type of mouse and also in any one region between germfree and conventionalized mice. As would be expected, there were fewer differences in amino acid distribution between the types of mice in both regions of the small intestine. This correlates with a much lower population of microorganisms in these regions. The changes in free amino acid content and distribution produced by microflora are great enough to serve as a good probe for studying the interactions of a limited number of species of microbes in gnotobiotic animals and assign possible specific functions to each species.

Substrate-protein interaction in tryptophanase from Bacillus alvei. Kinetic and spectral evaluations.

This investigation studied the substrate protein interaction of the alpha, beta elimination reaction in tryptophanase (EC 4.1.99.1). The results of this work are 2-fold. (a) The presence of multiple enzyme sites was found to be related to the observed kinetic patterns of inhibition. Indole analogues caused competitive inhibition in the tryptophanase reaction and noncompetitive inhibition in the dehydratase reaction. Inhibition patterns of alanine for these activities were reserved. (b) Under some conditions, compounds which bind presumably at the indole site modified the spectral and fluorescent characteristics of the enzyme. The addition of anthranilate to the enzyme resulted in a broad absorption band around 350 nm. This absorption band was distinct from that formed by alanine addition. Based on absorption data, both of these compounds could be bound simultaneously. The optical activity of tryptophanase was reported for the first time. Indole analogues caused greater conformational alterations in the circular dichroism spectra than 3-carbon analogues. The calculated anisotrophy factors, as well as fluorescent quenching data, suggest a more direct interaction between indole analogues and pyridoxal-P than between 3-carbon compounds that the coenzyme. It is proposed that the indole site is the dominant recognition site. The data are consistent with the three-dimensional aspects of space-filling models of Schiff's bases evaluated in terms of multiple site binding.

Pyridoxal 5'-phosphate and analogs as probes of coenzyme-protein interaction in Baccillus alvei tryptophanase.

Trytophanase from Bacillus alvei was resolved from its coenzyme, pyridoxal phosphate, by treatment with cysteine followed by column chromatography. Spectrophotometric titration of apoenzyme with pyridoxal-P showed 1 mol of pyridoxal-P bound per 52,000 g of enzyme. Kinetic analysis of coenzyme binding showed hyperbolic activation curves with a Km of 1.6 muM. Pyridoxal-P was used as a natural active site probe in spectrophotometric studies to distinguish differences in the active center of holotryptophanase and reconstituted enzyme that were not apparent by other techniques. The pKa for holotryptophanase is 7.9 while the pKa for reconstituted apoenzyme is 8.4. Apotryptophanase binds 2-nor, 2'-methyl, 2'-hydroxy, 6-methyl, and N-oxide pyridoxal-P to form analog enzymes distinguishable on the basis of absorption spectra and relative activity in catalyzing both the alpha, beta-elimination and beta-replacement reactions of tryptophanase. Apoenzyme also binds pyridoxal but pyridoxal analog enzyme is not active.

Structural role of pyridoxal 5'-phosphate, pyridoxal 5'-phosphate analogs, and other agents in the association of subunits of Bacillus alvei apotryptophanase.

Bacillus alvei apotryptophanase readily dissociates at low protein concentration and sediments at 5.7 S (dimer) in 0.01 M potassium phosphate (pH 7.8) from 9 to 33 degrees. With temperature held constant at 9 degrees, increasing the potassium, sodium, or ammonium phosphate buffer concentration increases the sedimentation value to 8.0 S. Increasing the monovalent cation concentration alone does not have the effect. Imidazole and pyridoxal compete with phosphate, preventing the effect. Raising the temperature to 26 degrees in the presence of high concentrations of potassium phosphate increases the sedimentation constant to 9.4 S. The addition of pyridoxal-P converts the dimer to a 9.4S tetramer. The conversion is dependent upon coenzyme concentration, temperature, and the nature of monovalent cation present. The Km for pyridoxal-P for the sodium form of the enzyme is more than tenfold greater than the Km for the potassium form of the enzyme. 2'-Methyl, 2'-hydroxyl, 6-methyl, and the N-oxide of pyridoxal-P are active in the association of dimer to tetramer but to differing extents. Analogs altered in the 4'-formyl position are also inactive structurally. Anthranilic acid, a competitive inhibitor of tryptophan, and 8-anilino-1-naphthalenesulfonic acid (ANS), a competitive inhibitor of pyridoxal-P binding, are both active in affecting the dimer to tetramer association but tryptophan is not. The dimer and tetramer are spectrally distinguishable through circular dichroic measurements, fluroescence quenching with pyridoxal-P or pyridoxal, and fluorescence enhancement with ANS. Pyridoxal-P causes the release of ANS from an ANS-apoenzyme complex.

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.

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.

Escherichia coli tryptophanase in the enteric environment.

The activity of the enzyme tryptophanase in the enteric environment was investigated to elucidate the significance of the enzyme in the metabolism of Escherichia coli. The tryptophanase activity, tryptophan content, and indole concentration as well as the numbers of E. coli were determined in the intestinal and fecal contents of conventional, germ-free, and monocontaminated axenic laboratory mice. Increasing the tryptophan content of the diet of mice having a conventional microflora increased the tryptophanase activity of the enteric microflora by a factor of almost 2 but did not increase the numbers of E. coli either absolutely or relative to other facultative enteric coliforms. In the enteric environment, E. coli is responsible for very little tryptophanase activity, a fraction calculated to be less than 0.02%. The values for the experimental parameters were much the same in the contents of the cecum and in the fecal material.

Tryptophanase-positive bacteria in the marine environment.

The prevalence of indole-positive organisms in the gut and environment of marine animals was studied. Indole formation by a group of the isolates was found to occur only in the presence of tryptophan. The isolates examined were all assigned to the genus Vibrio.

Physiological studies of biosynthetic indole excretion in Bacillus alvei.

Bacillus alvei excretes indole during early exponential growth in acid-hydrolyzed casein medium. l-Threonine is the amino acid responsible for "early" indole excretion, and the amount of indole excreted is directly related to the amount of l-threonine in the medium. "Early-indole" excretion can be prevented by the continuous addition of serine (3.1 mumoles per ml per hr) or by substituting a mutant with an impaired ability to degrade serine. The addition of serine to a culture during the period of indole excretion halts the excretion and stimulates indole utilization. Threonine is a competitive inhibitor of serine (K(i) = 0.6 m) in the tryptophan synthetase B reaction. The internal tryptophan concentration increases during the period of indole excretion, suggesting that threonine acts by increasing the activity of the tryptophan pathway. This view is supported by experiments demonstrating that anthranilic acid and indoleacrylic acid also stimulate indole excretion. A metabolic explanation is offered and discussed.

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.

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.

Physiological and kinetic studies with anthranilate synthetase of Bacillus alvei.

Anthranilate synthetase from Bacillus alvei was partially purified by ammonium sulfate fractionation and was stabilized by glycerol. The reaction mechanism of the enzyme was found to be sequential with respect to substrate, and the enzyme formed a hydroxamic acid in the absence of Mg(++). The K(m) for chorismic acid was 1.25 x 10(-4)m, and the K(m) for l-glutamine was 5.5 x 10(-4)m. Enzyme activity was inhibited by tryptophan noncompetitively with respect to chorismic acid and uncompetitively with respect to l-glutamine. An analysis of the inhibition patterns indicated that tryptophan may act as a dead end inhibitor and bind at the catalytic site. Enzyme activity could be completely inhibited in vitro and in vivo under the appropriate conditions, and enzyme synthesis was sensitive to repression by tryptophan. A sedimentation coefficient of 5.5S and an estimated molecular weight of 90,000 were obtained for the enzyme.

Tryptophanase in diverse bacterial species.

The distribution of tryptophanase was studied. The highest observed specific activity, mumoles per minute per milligram (dry weight) cells, is given in parentheses after each species. Tryptophanase was inducible and repressible in Escherichia coli (.914), Paracolobactrum coliforme (.210), Proteus vulgaris (.146), Aeromonas liquefaciens (.030), Photobacterium harveyi (.035), Sphaerophorus varius (.021), Bacteroides sp. (.048), and Corynebacterium acnes (.042). The enzyme was constitutive and nonrepressible in Bacillus alvei (.013), and was inducible but not repressible by glucose in Micrococcus aerogenes (.036). Indole-positive bacteria were found in fecal or intestinal samples from a variety of animals among the mammals, reptiles, insects, molluscs, fish, crustaceans, and amphibians.

N-Acetylation of glucosamine-6-phosphate in Leuconostoc mesenteroides.

A partially purified enzyme (120-fold) from Leuconostoc mesenteroides catalyzed the reversible N-acetylation of d-glucosamine-6-phosphate. Coenzyme A was not required and inhibited the reaction rate. Neither d-glucosamine nor N-acetyl-d-glucosamine served as a substrate for the reversible reaction. The enzyme preparation retained 50% of its original activity after 5 min at 100 C. The K(m) for acetate was 7.7 x 10(-2)m in the presence of 2 x 10(-2)md-glucosamine-6-phosphate. The K(m) for d-glucosamine-6-phosphate was 5.0 x 10(-3)m in the presence of 0.64 m acetate. The product of the reaction was characterized by comparison with N-acetyl-d-glucosamine-6-phosphate prepared by enzymatic phosphorylation of N-acetyl-d-glusamine. The characterization tests were: chromatographic migration, acid hydrolysis, enzymatic dephosphorylation, sodium borohydride reduction, and periodate oxidation. The equilibrium constant for the reaction was about 7.5 m for the expression K = (d-glucosamine-6-phosphate)(acetate)/N-acetyl-d-glucosamine-6-phosphate. The standard free energy of the reaction was approximately 1,200 cal per mole.

Kinases in Leuconostoc mesenteroides.

Enzyme extracts of Leuconostoc mesenteroides were found to contain at least four separate kinases: one active with glucose, glucosamine, and N-acetylglucosamine; one active with fructose and mannose; and two active with gluconate, one constitutive and one inducible. The molecular sizes of all the kinases, estimated from sucrose gradient centrifugation, are about the same, 37,000 to 50,000 daltons, except the constitutive gluconate kinase, which is about 100,000 daltons. Apparent Michaelis constants were calculated for all of the substrates mentioned. The kinases are separable on triethylaminoethyl cellulose.