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PubMed1978

Stereochemistry and mechanism of reactions catalyzed by tryptophanase Escherichia coli.

Several beta replacement and alpha,beta elimination reactions catalyzed by tryptophanase from Escherichia coli are shown to proceed stereospecifically with retention of configuration. These conversions include synthesis of tryptophan from (2S,3R)- and (2s,3s)-[3(-3H)]serine in the presence of indole, deamination of these serines in D2O to pyruvate and ammonia, and cleavage of (2S,3R)-and (2S,3S)-[3(-3H)]tryptophan in D2O to indole, pyruvate, and ammonia. A coupled reaction with lactate dehydrogenase was used to trap the stereospecifically labeled [3-H,2H,3H]pryuvates as lactate, which was oxidized to acetate for chirality analysis of the methyl group. During deamination of tryptophan there is significant intramolecular transfer of the alpha proton of the amino acid to C-3 of indole. To determine the exposed face of the cofactor.substrate complex on the enzyme surface and to analyze its conformational orientation, sodium boro[3H]hydride was used to reduce tryptophanase-bound alaninepyridoxal phosphate Schiff's base. Degradation of the resulting pyridoxylalanine to (2S)-[2(-3H)]alanine and (4'S)-[4'(-3H)]pyridoxamine demonstrates that reduction occurs from the exposed si face at C-4' of the complex and that the ketimine double bond is trans.

Carbon Radioisotopes↗

PubMed1983

Phosphorus 31 nuclear magnetic resonance study of tryptophanase. Pyridoxal phosphate-binding site.

The pyridoxal phosphate-dependent enzyme tryptophanase has been investigated using 31P nuclear magnetic resonance at 72.86 MHz. In the native enzyme, the pyridoxal-P 31P chemical shift was found to be 3.55 ppm and independent of pH, indicating that the dianionic phosphate group of the cofactor is not accessible to solvent. Binding of the competitive inhibitor, beta-phenyl-DL-serine, results in the formation of the transaldimination complex. This complex is fixed to the enzyme via the dianionic phosphate group of the cofactor: again the observed shift is independent of pH. In both cases, restricted rotational freedom of the phosphate group around the C-O bond linking the phosphate ester to the pyridine moiety of the cofactor could be asserted from line width data. Addition of the competitive inhibitor, L-alanine, to tryptophanase produces the quinonoid intermediate. The phosphate group of this complex has lost its specific interaction (probably a salt bridge) with the protein, as indicated by the pH dependence of the chemical shift.

Binding Sites↗

PubMed1972

Site of action of 3',5'-cyclic adenosine monophosphate in production of tryptophanase in Escherichia coli.

3',5' cyclic-AMP (cAMP) will stimulate the rate of tryptophanase synthesis in Escherichia coli cultures induced with tryptophan. Adding cAMP after the initiation of messenger RNA synthesis was blocked by rifampicin, did not stimulate tryptophanase synthesis. This indicates that cAMP acts at initiation of either transcription or translation and not at the level of chain elongation of either the messenger or the polypeptide chain.

Cyclic AMP↗

PubMed1969

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.

Animals↗

PubMed2000

Activation of the coenzyme at the early step of the catalytic cycle of tryptophanase.

Tryptophanase is catalytically more competent in alkaline pH even though the coenzyme exists as an inactive aldamine structure in this pH region. The binding of a substrate analog, 3-indolepropionate to the enzyme shifts the equilibrium from the substituted aldamine to the ketoenamine form in the entire pH region studied. The resultant ketoenamine form is favorable for transaldimination with the substrate amino group, a prerequisite for subsequent catalysis. This implies that the binding of tryptophan in alkaline pH, where the enzyme shows maximum activity, converts the inactive aldamine form of the coenzyme to the active ketoenamine form, which is favorable for undergoing the next step of the catalytic process.

Enzyme Activation↗

PubMed1998

Crystal structure of tryptophanase.

The X-ray structure of tryptophanase (Tnase) reveals the interactions responsible for binding of the pyridoxal 5'-phosphate (PLP) and atomic details of the K+ binding site essential for catalysis. The structure of holo Tnase from Proteus vulgaris (space group P2(1)2(1)2(1) with a = 115.0 A, b = 118.2 A, c = 153.7 A) has been determined at 2.1 A resolution by molecular replacement using tyrosine phenol-lyase (TPL) coordinates. The final model of Tnase, refined to an R-factor of 18.7%, (Rfree = 22.8%) suggests that the PLP-enzyme from observed in the structure is a ketoenamine. PLP is bound in a cleft formed by both the small and large domains of one subunit and the large domain of the adjacent subunit in the so-called "catalytic" dimer. The K+ cations are located on the interface of the subunits in the dimer. The structure of the catalytic dimer and mode of PLP binding in Tnase resemble those found in aspartate amino-transferase, TPL, omega-amino acid pyruvate aminotransferase, dialkylglycine decarboxylase (DGD), cystathionine beta-lyase and ornithine decarboxylase. No structural similarity has been detected between Tnase and the beta 2 dimer of tryptophan synthase which catalyses the same beta-replacement reaction. The single monovalent cation binding site of Tnase is similar to that of TPL, but differs from either of those in DGD.

Amino Acid Sequence↗

PubMed1991

Application of the tryptophanase promoter to high expression of the tryptophan synthase gene in Escherichia coli.

The application of an inducible regulation system using the tryptophanase operon promoter (TPase promoter; Ptna) was examined for its high expression of the tryptophan synthase (TS) gene in Escherichia coli. The main problem in the application of Ptna for industrial purposes is catabolite repression by glucose, since glucose is the most abundant carbon source. However, this problem could be avoided by changing glucose to an organic acid, such as succinate, fumarate, malate and acetate, in the course of cultivation after glucose initially added was completely consumed. Under these conditions, L-tryptophan was also used to induce tryptophan synthase. Thus, the specific activity of TS in E. coli strain no. 168 harbouring pBR322F-Ptna TS was increased 500-fold compared to that of the cultured host strain. About 1 mol L-tryptophan/l reaction mixture was formed from indole and L-serine at 37 degrees C for 3.5 h.

Carbon↗

PubMedDate not supplied

Effect of environmental toxicants on enzyme biosynthesis: a comparison of beta-galactosidase, alpha-glucosidase and tryptophanase.

Except for beta-galactosidase, little is known about the effect of environmental toxicants on enzyme induction. The information could be potentially useful for the development of low-cost and rapid ecotoxicity assays. The effect of toxicants on the de novo biosynthesis of three inducible enzymes, beta-galactosidase and tryptophanase in E. coli and alpha-glucosidase in B. subtilis was investigated. Biosynthesis of alpha-glucosidase was the most sensitive to environmental toxicants, particularly pentachlorophenol and sodium dodecyl sulfate. The sensitivity of B. subtilis to toxicants was further increased when Tween 80 was incorporated in the growth medium.

Bacteria↗

PubMed1992

Synthesis of serine-AMC-carbamate: a fluorogenic tryptophanase substrate.

A new fluorogenic substrate for the pyridoxal 5'-phosphate-dependent enzyme tryptophanase is described. L-Serine, which is linked to 7-amino-4-methylcoumarin through an O-carbamoyl tether, serves as a substrate for the enzyme. The released moiety, 7-amino-4-methylcoumarin (AMC), can be detected by either absorbance (355 nm) or fluorescence (excitation 365 nm/emission 440 nm). Kinetic constants were measured using each of these techniques: Km = 85 +/- 20 microM, Vmax = 2.9 +/- 0.4 mumol/min/mg (fluorescence) and Km = 129 +/- 21 microM, Vmax = 3.1 +/- 0.3 mumol/min/mg (absorbance). The Vmax for serine-AMC-carbamate is approximately 1.9 times faster than that of the natural substrate, tryptophan. Using fluorescence detection, solutions containing 10(-3) units of activity could be routinely assayed.

Coumarins↗

PubMed1975

The tryptophanase from Proteus rettgeri, improved purification and properties of crystalline holotryptophanase.

The inducible tryptophanase (L-tryptophan indole-lyase (deaminating) EC 4.1.99.1) was crystallized in holoenzyme from the cell extract of Proteus rettgeri. The purification procedure included ammonium sulfate fractionation, heat treatment at 60 degrees C, DEAE-Sephadex and hydroxylapatite column chromatographies. Crystallization was performed by the addition of ammonium sulfate to the purified enzyme solution containing 20% (v/v) glycerol, 0.1 mM pyridoxal phosphate and 10 mM mercaptoethanol. The crystallized enzyme was yellow and showed absorption maxima at 340 and 420 nm. The crystalline holotryptophanase preparation was homogeneous by the criteria of ultracentrifugation and disc gel electrophoresis. The molecular weight of the enzyme was calculated as approx. 222 000. The amount of pyridoxal phosphate bound to the enzyme was determined to be 4 mol per mol of the enzyme. The enzyme is composed of four subunits of identical molecular size (mol. wt 55 000) and irreversibly dissociates into these subunits in the presence of a high concentration of sodium dodecylsulfate or guanidine hydrochloride. The NH2-terminal amino acid of the enzyme was identified as alanine.

Amino Acid Sequence↗

PubMed1986

Catalytic function of a tyrosyl residue in tryptophanase.

Tryptophanase has an essential tyrosyl residue/active site which can be modified by tetranitromethane. Pyridoxal 5'-phosphate can prevent this modification efficiently, whereas pyridoxal 5'-phosphate N-oxide cannot, indicating that the free pyridinium N is required for the interaction of the coenzyme with the tyrosyl residue, probably via a hydrogen bond. The weakened binding of the coenzyme to the modified enzyme was confirmed on gel filtration, the modified enzyme being dissociated from the coenzyme seven-fold faster than the native enzyme. Furthermore, absorption spectral analyses demonstrated that the modified enzyme can catalyze the transaldimination step, but fails to abstract the alpha-H of substrates. The tyrosyl residue, therefore, not only participates in coenzyme binding, but also contributes to alpha-H labilization.

Binding Sites↗

PubMed1991

Preliminary X-ray crystallographic analysis of tryptophanase from Escherichia coli.

Tryptophanase (L-tryptophan indole-lyase) from Escherichia coli has been crystallized from ammonium sulfate solution using a vapor diffusion method. The crystals are tetragonal and belong to space group P4(1)2(1)2 or its enantiomorph. The cell dimensions of the crystals are a = b = 113.4 A, and c = 232.2 A, with two subunits per asymmetric unit. The crystals diffract to at least 3 A resolution, and are suitable for X-ray structural analysis.

Ammonium Sulfate↗

PubMed1996

Interactions of Escherichia coli tryptophanase with quasisubstrates and monovalent cations studied by the circular dichroism and fluorescence methods.

The reaction of tryptophanase and its W330F and W248F mutant forms with quasi-substrates forming an external pyridoxal phosphate aldimine or quinonoid is accompanied by the appearance of a positive circular dichroism (CD) peak at 290 nm. The peak seems to arise from a Tyr residue undergoing reorientation during the reaction. The peak does not appear upon formation of non-covalent Michaelis complexes of the enzyme with quasi-substrates such as indolepropionate, beta-phenyllactate and alpha-methylphenylalanine. The non-covalent complexes and external aldimines exhibit similar absorption spectra but can be distinguished by their CD and by the intensity of their fluorescence. Formation of the non-covalent complexes leads to an increase in positive CD at 420 nm while formation of the external aldimines leads to disappearance of the positive CD at 420 nm and its replacement by negative CD; it also leads to strong quenching of the coenzyme fluorescence at 500 nm. The quantum yield of fluorescence of the external aldimines is 6-times lower than that of the internal aldimine. Activating cations (K+, NH4+) strongly diminish the intensity of a negative protein CD band at 275 nm. From a comparison of the intensity of this band in the spectra of the wild-type holo- and apoenzyme and in the tryptophan mutants, it was deduced that the band belongs to a Tyr residue, which may be a part of the cation-binding site or located in its immediate vicinity.

Binding Sites↗

PubMed2006

Tryptophanase from Proteus vulgaris: the conformational rearrangement in the active site, induced by the mutation of Tyrosine 72 to phenylalanine, and its mechanistic consequences.

Tyr72 is located at the active site of tryptophanase (Trpase) from Proteus vulgaris. For the wild-type Trpase Tyr72 might be considered as the general acid catalyst at the stage of elimination of the leaving groups. The replacement of Tyr72 by Phe leads to a decrease in activity for L-tryptophan by 50,000-fold and to a considerable rearrangement of the active site of Trpase. This rearrangement leads to an increase of room around the alpha-C atom of any bound amino acid, such that covalent binding of alpha-methyl-substituted amino acids becomes possible (which cannot be realized in wild-type Trpase). The changes in reactivities of S-alkyl-L-cysteines provide evidence for an increase of congestion in the proximity of their side groups in the mutant enzyme as compared to wild-type enzyme. The observed alteration of catalytic properties in a large degree originates from a conformational change in the active site. The Y72F Trpase retains significant activity for L-serine, which allowed us to conclude that in the mutant enzyme, some functional group is present which fulfills the role of the general acid catalyst in reactions associated with elimination of small leaving groups.

Amino Acid Sequence↗

PubMed1990

Role of tryptophan 248 in the active site of tryptophanase from Escherichia coli.

Tryptophan 248, located in the active site of tryptophanase from Escherichia coli, has been replaced with phenylalanine by site-directed mutagenesis. Judging from CD and fluorescence spectra, the global structure of the mutant enzyme was found to be the same as that of the wild-type enzyme. The binding affinity of the mutant enzyme for the coenzyme pyridoxal 5'-phosphate (PLP) was reduced tenfold compared to the wild-type enzyme. Kinetic analyses under PLP-saturated conditions indicated that the Km values of the mutant enzyme for substrates are the same as those of wild-type enzyme but the kcat values are decreased to about 85%, which accounts for the overall activity decrease. These findings suggest that tryptophan 248 interacts closely with PLP and plays an important role in the catalytic reaction.

Animals↗

PubMed2002

Cold-induced enzyme inactivation: how does cooling lead to pyridoxal phosphate-aldimine bond cleavage in tryptophanase?

The phenomenon of cold scission or cold lability, which entails a widespread variety of oligomeric enzymes, is still enigmatic. The effect of cooling on the activity and the quaternary structure of the pyridoxal 5'-phosphate (PLP)-dependent enzyme, tryptophanase (Tnase), was studied utilizing single photon counting time-resolved spectrofluorometry. Upon cooling of holo-wild-type (wt) Tnase and its W330F mutant from 25 degrees C to 2 degrees C, a reduction in PLP fluorescence lifetime and rotational correlation time as well as inactivation and dissociation from tetramers to dimers were observed for both enzymes. Fluorescence anisotropy invariably decreased as a consequence of cooling, whether it was accompanied by a slight decrease in activity without significant dissociation, or by a substantial decrease in activity that was associated with either a partial or major dissociation. These results support the suggested conformational change that precedes the PLP-aldimine bond scission. It is proposed that cold inactivation is initiated by the weakening of hydrophobic interactions, leading to conformational changes which are the driving force for the aldimine bond cleavage.

Anisotropy↗

PubMed1981

Kinetics of pH-dependent interconversion of tryptophanase spectral forms studied by scanning stopped-flow spectrophotometry.

Morino and Snell [Morino, Y., & Snell, E. E. (1967) J. Biol. Chem. 242, 5591-5601] previously showed that the relative amplitudes of the 337- and 420-nm absorption bands of tryptophanase depended on both pH and the nature of a required monovalent cation activator. An investigation of the kinetics of interconversion of the 337- and 420-nm forms following a rapid incremental increase (jump) or decrease (drop) in pH over the range of enzyme stability in 0.2 M KCl at 24 +/- 0.3 degrees C by scanning stopped-flow spectrophotometry showed three distinct time-dependent phases. They were (1) an abrupt phase which is complete in less than 6.5 ms, (2) a fast first-order interconversion of the 420- and 337-nm absorbances, and (3) a slow first-order process involving growth at 355 nm coupled to two decays centered at 325 and 430 nm in the incremental pH jumps and decay at 355 nm with concomitant growth at 430 and 290 nm in the incremental pH-drop experiments. The results of these experiments were analyzed in terms of a scheme involving enzyme forms E alpha, E beta, E beta H+, E gamma, E gamma H+, and E delta. The E alpha form predominates in the absence of activating monovalent cations and absorbs at 420 nm. Those in the beta manifold, E beta and E beta H+, also absorb at 420 nm while those in the gamma manifold, E gamma and E gamma H+, absorb at 337 nm. The form E delta absorbs at 335 nm. E beta H+ and E gamma H+ represent the protonated form of the enzyme in each manifold. Analysis of the abrupt phase showed no significant systematic changes in absorbance above 330 nm for either the pH-jump or pH-drop experiments. The fast second phase involves the first-order interconversion of the beta and gamma manifolds while the slow third phase describes the buildup or decay of the delta manifold. Presumably conformational changes control the rate of these interconversions. The pH dependence of the fast first-order beta to gamma conversion was described and evaluated in terms of five independent equilibrium and rate constants and three independent amplitude terms by simultaneously fitting the amplitude data and first-order rate constants to an equation describing the overall scheme with a nonlinear least-squares program KINFIT4 [Dye, J. L., & Nicely, V. A. (1971) J. Chem. Educ. 48, 443-448]. The pK for protonation of the beta form = 9.70 +/- 0.12, for protonation of the gamma form (337-nm absorber) = 6.77 +/- 0.10, and for the pH-dependent interconversion of the beta and gamma manifolds, pKa = 8.11 +/- 0.04. The computed equilibrium distribution among the four species of the beta and gamma manifolds showed that E beta H+ and E gamma predominate.

Drug Stability↗

PubMed1975

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.

Apoenzymes↗