Tryptophan Indole-lyase Research Articles

New tryptophanase inhibitors: Towards prevention of bacterial biofilm formation

Tryptophanase (tryptophan indole-lyase, Tnase, EC 4.1.99.1), a bacterial enzyme with no counterpart in eukaryotic cells, produces from L-tryptophan pyruvate, ammonia and indole. It was recently suggested that indole signaling plays an important role in the stable maintenance of multicopy plasmids. In addition, Tnase was shown to be capable of binding Rcd, a short RNA molecule involved in resolution of plasmid multimers. Binding of Rcd increases the affinity of Tnase for tryptophan, and it was proposed that indole is involved in bacteria multiplication and biofilm formation. Biofilm-associated bacteria may cause serious infections, and biofilm contamination of equipment and food, may result in expensive consequences. Thus, optimal and specific factors that interact with Tnase can be used as a tool to study the role of this multifunctional enzyme as well as antibacterial agents that may affect biofilm formation. Most known quasi-substrates inhibit Tnase at the mM range. In the present work, the mode of Tnase inhibition by the following compounds and the corresponding Ki values were: S-phenylbenzoquinone-L-tryptophan, uncompetitively, 101 μM; α-amino-2-(9,10-anthraquinone)-propanoic acid, noncompetitively, 174 μM; L-tryptophane-ethylester, competitively, 52 μM; N-acetyl-L-tryptophan, noncompetitively, 48 μM. S-phenylbenzoquinone-L-tryptophan and α-amino-2-(9,10-anthraquinone)-propanoic acid were newly synthesized.

Hydrostatic Pressure as a Structural and Mechanistic Probe of Tryptophan Synthase and Tryptophan Indole‐lyase

The effects of hydrostatic pressure on the conformation and activity of tryptophan (Trp) synthase α2β2 complex from Salmonella typhimurium and tryptophan indole-lyase (Trpase) from Escherichia coli have been examined. The data demonstrate that the open conformation of Trp synthase is favored by higher pressure. The ΔVo and Ko for the equilibrium between the closed and open conformations of the Trp synthase-L-Ser complex are −126 mL/mol and 0.12 for the Na+form and −171 mL/mol and 2.3 x 10−4 for the NH4+ form. Pressure-jump experiments of the H463F mutant Trpase L-Trp and L-Met complexes show relaxations at 500 nm after rapid pressure changes of 100–400 bar for the complexes with both L-Trp and L-Met. However, the apparent rate constants for relaxation of L-Trp, but not L-Met, show a significant increase with pressure. These results suggest that there may be a contribution to the deprotonation reaction of Trpase either from quantum mechanical tunneling or from a mechanical coupling of protein motion and proton transfer for the reaction of L-Trp, but not with L-Met.

Differential Effects of Temperature and Hydrostatic Pressure on the Formation of Quinonoid Intermediates from l-Trp and l-Met by H463F Mutant Escherichia coli Tryptophan Indole-lyase

Escherichia coli tryptophan indole-lyase (Trpase) is a bacterial pyridoxal 5'-phosphate (PLP)-dependent enzyme which catalyzes the reversible beta-elimination of l-Trp to give indole and ammonium pyruvate. H463F mutant E. coli Trpase (H463F Trpase) has very low activity with l-Trp, but it has near wild-type activity with other in vitro substrates, such as S-ethyl-l-cysteine and S-(o-nitrophenyl)-l-cysteine [Phillips, R. S., Johnson, N., and Kamath, A. V. (2002) Formation in vitro of Hybrid Dimers of H463F and Y74F Mutant Escherichia coli Tryptophan Indole-lyase Rescues Activity with l-Tryptophan, Biochemistry 41, 4012-4019]. The interaction of H463F Trpase with l-Trp and l-Met, a competitive inhibitor, has been investigated by rapid-scanning stopped-flow, high-pressure, and pressure jump spectrophotometry. Both l-Trp and l-Met bind to H463F Trpase to form equilibrating mixtures of external aldimine and quinonoid intermediates, absorbing at approximately 420 and approximately 505 nm, respectively. The apparent rate constant for quinonoid intermediate formation exhibits a hyperbolic dependence on l-Trp and l-Met concentration. The rate constant for quinonoid intermediate formation from l-Trp is approximately 10-fold lower for H463F Trpase than for wild-type Trpase, but the rate constant for reaction of l-Met is similar for H463F Trpase and wild-type Trpase. The temperature dependence of the rate constants for quinonoid intermediate formation reveals that both l-Trp and l-Met have similar values of DeltaH(++), but l-Met has a more negative value of DeltaS(++). Hydrostatic pressure perturbs the spectra of the H463F l-Trp and l-Met complexes, by shifting the position of the equilibria between different quinonoid and external aldimine complexes. Pressure-jump experiments show relaxations at 500 nm after rapid pressure changes of 100-400 bar with both l-Trp and l-Met. The apparent rate constants for relaxation of l-Trp, but not l-Met, show a significant increase with pressure. From these data, the value of DeltaV(++) for quinonoid intermediate formation from the external aldimine of l-Trp can be estimated to be -26.5 mL/mol, a larger than expected negative value for a proton transfer. These results suggest that there may be a contribution to the deprotonation reaction either from quantum mechanical tunneling or from a mechanical coupling of protein motion and proton transfer associated with the reaction of l-Trp, but not with l-Met.

Role of Lysine-256 in Citrobacter freundii Tyrosine Phenol-lyase in Monovalent Cation Activation

Tyrosine phenol-lyase (TPL) from Citrobacter freundii is dependent on monovalent cations, K(+) or NH(4)(+), for high activity. We have shown previously that Glu-69, which is a ligand to the bound cation, is important in monovalent cation binding and activation [Sundararaju, B., Chen, H., Shillcutt, S., and Phillips, R. S. (2000) Biochemistry 39, 8546-8555]. Lys-256 is located in the monovalent cation binding site of TPL, where it forms a hydrogen bond with a structural water bound to the cation. This lysine residue is highly conserved in sequences of TPL and the paralogue, tryptophan indole-lyase. We have now prepared K256A, K256H, K256R, and E69D/K256R mutant TPLs to probe the role of Lys-256 in monovalent cation binding and activation. K256A and K256H TPLs have low activity (k(cat)/K(m) values of 0.01-0.1%), are not activated by monovalent cations, and do not exhibit fluorescence emission at 500 nm from the PLP cofactor. In contrast, K256R TPL has higher activity (k(cat)/K(m) about 10% of wild-type TPL), is activated by K(+), and exhibits fluorescence emission from the PLP cofactor. K256A, K256H, and K256R TPLs bind PLP somewhat weaker than wild-type TPL. E69D/K256R TPL was prepared to determine if the guanidine side chain could substitute for the monovalent cation. This mutant TPL has wild-type activity with S-Et-L-Cys or S-(o-nitrophenyl)-L-Cys but has no detectable activity with L-Tyr. E69D/K256R TPL is not activated by monovalent cations and does not show PLP fluorescence. In contrast to wild-type and other mutant TPLs, PLP binding to E69D/K256R is very slow, requiring several hours of incubation to obtain 1 mol of PLP per subunit. Thus, E69D/K256R TPL appears to have altered dynamics. All of the mutant TPLs react with inhibitors, L-Ala, L-Met, and L-Phe, to form equilibrating mixtures of external aldimine and quinonoid intermediates. Thus, Lys-256 is not the base which removes the alpha-proton during catalysis. The results show that the function of Lys-256 in TPL is in monovalent cation binding and activation.

Tyrosine phenol-lyase and tryptophan indole-lyase encapsulated in wet nanoporous silica gels: Selective stabilization of tertiary conformations.

The pyridoxal 5'-phosphate-dependent enzymes tyrosine phenol-lyase and tryptophan indole-lyase were encapsulated in wet nanoporous silica gels, a powerful method to selectively stabilize tertiary and quaternary protein conformations and to develop bioreactors and biosensors. A comparison of the enzyme reactivity in silica gels and in solution was carried out by determining equilibrium and kinetic parameters, exploiting the distinct spectral properties of catalytic intermediates and reaction products. The encapsulated enzymes exhibit altered distributions of ketoenamine and enolimine tautomers, increased values of inhibitors dissociation constants, slow attaining of steady-state in the presence of substrate and substrate analogs, modified steady-state distribution of catalytic intermediates, and a sixfold-eightfold decrease of specific activities. This behavior can be rationalized by a reduced conformational flexibility for the encapsulated enzymes and a selective stabilization of either the open (inactive) or the closed (active) form of the enzymes. Despite very similar structures and catalytic mechanisms, the influence of encapsulation is more pronounced for tyrosine phenol-lyase than tryptophan indole-lyase. This finding indicates that subtle structural and dynamic differences can lead to distinct interactions of the protein with the gel matrix.

Role of arginine 226 in the mechanism of tryptophan indole-lyase from Proteus vulgaris.

In the spatial structure of tryptophanase from Proteus vulgaris the guanidinium group of arginine 226 forms a salt bridge with the 3;-oxygen atom of the coenzyme. The replacement of arginine 226 with alanine using site-directed mutagenesis reduced the affinity of the coenzyme for the protein by one order of magnitude compared to the wild-type enzyme. The catalytic activity of the mutant enzyme in the reaction with L-tryptophan was reduced 10(5)-fold compared to the wild-type enzyme. The rates of the reactions with some other substrates decreased 10(3)-10(4)-fold. The mutant enzyme catalyzed exchange of the C-alpha-proton in complexes with some inhibitors with rates reduced 10(2)-fold compared to the wild-type enzyme. Absorption and circular dichroism spectra of the mutant enzyme and the enzyme-inhibitor complexes demonstrate that the replacement of arginine 226 with alanine does not significantly affect the tautomeric equilibrium of the internal aldimine, but it leads to an alteration of the optimal conformation of the coenzyme-substrate intermediates.

Role of Aspartate-133 and Histidine-458 in the Mechanism of Tryptophan Indole-Lyase from Proteus vulgaris

Tryptophan indole-lyase (Trpase) from Proteus vulgaris is a pyridoxal 5'-phosphate dependent enzyme that catalyzes the reversible hydrolytic cleavage of L-Trp to yield indole and ammonium pyruvate. Asp-133 and His-458 are strictly conserved in all sequences of Trpase, and they are located in the proposed substrate-binding region of Trpase. These residues were mutated to alanine to probe their role in substrate binding and catalysis. D133A mutant Trpase has no measurable activity with L-Trp as substrate, but still retains activity with S-(o-nitrophenyl)-L-cysteine, S-alkyl-L-cysteines, and beta-chloro-L-alanine. H458A mutant Trpase has 1.6% of wild-type Trpase activity with L-Trp, and high activity with S-(o-nitrophenyl)-L-cysteine, S-alkyl-L-cysteines, and beta-chloro-L-alanine. H458A mutant Trpase does not exhibit the pK(a) of 5.3 seen in the pH dependence of k(cat)/K(m) of L-Trp for wild-type Trpase. Both mutant enzymes are inhibited by L-Ala, L-Met, and L-Phe, with K(i) values similar to those of wild-type Trpase, but oxindolyl-L-alanine and beta-phenyl-DL-serine show much weaker binding to the mutant enzymes, suggesting that Asp-133 and His-458 are involved in the binding of these ligands. D133A and H458A mutant Trpase exhibit absorption and CD spectra in the presence of substrates and inhibitors that are similar to wild-type Trpase, with peaks at about 420 and 500 nm. The rate constants for formation of the 500 nm bands for the mutant enzymes are equal to or greater than those of wild-type Trpase, indicating that Asp-133 and His-458 do not play a role in the formation of quinonoid intermediates. In constrast to wild-type and H458A mutant Trpase, D133A mutant Trpase forms an intermediate from S-ethyl-L-Cys that absorbs at 345 nm, and is likely to be an alpha-aminoacrylate. Crystals of D133A and H458A mutant Trpase bind amino acids with similar affinity as the proteins in solution, except for L-Ala, which binds to D133A mutant Trpase crystals about 20-fold stronger than in solution. These results suggest that Asp-133 and His-458 play an important role in the elimination reaction of L-Trp. Asp-133 likely forms a hydrogen bond directly to the indole NH of the substrate, while His-458 probably is hydrogen bonded to Asp-133.

Crystals of Tryptophan Indole-Lyase and Tyrosine Phenol-Lyase Form Stable Quinonoid Complexes

The binding of substrates and inhibitors to wild-type Proteus vulgaris tryptophan indole-lyase and to wild type and Y71F Citrobacter freundii tyrosine phenol-lyase was investigated in the crystalline state by polarized absorption microspectrophotometry. Oxindolyl-lalanine binds to tryptophan indole-lyase crystals to accumulate predominantly a stable quinonoid intermediate absorbing at 502 nm with a dissociation constant of 35 microm, approximately 10-fold higher than that in solution. l-Trp or l-Ser react with tryptophan indole-lyase crystals to give, as in solution, a mixture of external aldimine and quinonoid intermediates and gem-diamine and external aldimine intermediates, respectively. Different from previous solution studies (Phillips, R. S., Sundararju, B., & Faleev, N. G. (2000) J. Am. Chem. Soc. 122, 1008-1114), the reaction of benzimidazole and l-Trp or l-Ser with tryptophan indole-lyase crystals does not result in the formation of an alpha-aminoacrylate intermediate, suggesting that the crystal lattice might prevent a ligand-induced conformational change associated with this catalytic step. Wild-type tyrosine phenol-lyase crystals bind l-Met and l-Phe to form mixtures of external aldimine and quinonoid intermediates as in solution. A stable quinonoid intermediate with lambda(max) at 502 nm is accumulated in the reaction of crystals of Y71F tyrosine phenol-lyase, an inactive mutant, with 3-F-l-Tyr with a dissociation constant of 1 mm, approximately 10-fold higher than that in solution. The stability exhibited by the quinonoid intermediates formed both by wild-type tryptophan indole-lyase and by wild type and Y71F tyrosine phenol-lyase crystals demonstrates that they are suitable for structural determination by x-ray crystallography, thus allowing the elucidation of a key species of pyridoxal 5'-phosphate-dependent enzyme catalysis.

Threonine-124 and phenylalanine-448 in Citrobacter freundii tyrosine phenol-lyase are necessary for activity with L-tyrosine.

Thr-124 and Phe-448 are located in the active site of Citrobacter freundii tyrosine phenol-lyase (TPL) near the phenol ring of a bound substrate analogue, 3-(4'-hydroxyphenyl)propionic acid [Sundararaju, Antson, Phillips, Demidkina, Barbolina, Gollnick, Dodson and Wilson (1997) Biochemistry 36, 6502-6510]. Thr-124 is replaced by Asp and Phe-448 is replaced by His in the crystal structure of a structurally similar enzyme, Proteus vulgaris tryptophan indole-lyase, which has 50% identical residues. Hence, Thr-124 and Phe-448 in TPL were mutated to Ala or Asp, and His, respectively, in order to probe the role of these residues in the reaction specificity for L-Tyr. These mutant enzymes have little or no beta-elimination activity with L-Tyr or 3-fluoro-L-Tyr as a substrate, but retain significant elimination activity with S-(o-nitrophenyl)-L-cysteine, S-alkyl-L-cysteines and beta-chloroalanine. Furthermore, the binding of L-Tyr and other non-substrate amino acids is not significantly affected by the mutations. The mutant TPLs form intermediates in rapid-scanning stopped-flow experiments with L-Phe, L-Tyr and L-Trp, similar to those seen with wild-type TPL. These results demonstrate that Thr-124 and Phe-448 are necessary for the reaction specificity of TPL for L-Tyr, and probably play a role in the elimination stage of the reaction mechanism. Thr-124 is within hydrogen-bonding distance of the phenolic group of the bound substrate, and may help to orientate the ring for beta-elimination to occur. Phe-448 may be important to allow the formation of the closed conformation during the reaction.

Threonine-124 and phenylalanine-448 in Citrobacter freundii tyrosine phenol-lyase are necessary for activity with l-tyrosine

Thr-124 and Phe-448 are located in the active site of Citrobacter freundii tyrosine phenol-lyase (TPL) near the phenol ring of a bound substrate analogue, 3-(4′-hydroxyphenyl)propionic acid [Sundararaju, Antson, Phillips, Demidkina, Barbolina, Gollnick, Dodson and Wilson (1997) Biochemistry 36, 6502–6510]. Thr-124 is replaced by Asp and Phe-448 is replaced by His in the crystal structure of a structurally similar enzyme, Proteus vulgaris tryptophan indole-lyase, which has 50% identical residues. Hence, Thr-124 and Phe-448 in TPL were mutated to Ala or Asp, and His, respectively, in order to probe the role of these residues in the reaction specificity for l-Tyr. These mutant enzymes have little or no β-elimination activity with l-Tyr or 3-fluoro-l-Tyr as a substrate, but retain significant elimination activity with S-(o-nitrophenyl)-l-cysteine, S-alkyl-l-cysteines and β-chloroalanine. Furthermore, the binding of l-Tyr and other non-substrate amino acids is not significantly affected by the mutations. The mutant TPLs form intermediates in rapid-scanning stopped-flow experiments with l-Phe, l-Tyr and l-Trp, similar to those seen with wild-type TPL. These results demonstrate that Thr-124 and Phe-448 are necessary for the reaction specificity of TPL for l-Tyr, and probably play a role in the elimination stage of the reaction mechanism. Thr-124 is within hydrogen-bonding distance of the phenolic group of the bound substrate, and may help to orientate the ring for β-elimination to occur. Phe-448 may be important to allow the formation of the closed conformation during the reaction.

Formation in vitro of hybrid dimers of H463F and Y74F mutant Escherichia coli tryptophan indole-lyase rescues activity with L-tryptophan.

Y74F and H463F mutant forms of Escherichia coli tryptophan indole-lyase (Trpase) have been prepared. These mutant proteins have very low activity with L-Trp as substrate (kcat and kcat/Km values less than 0.1% of wild-type Trpase). In contrast, these mutant enzymes exhibit much higher activity with S-(o-nitrophenyl)-L-cysteine and S-ethyl-L-cysteine (kcat/Km values about 1-50% of wild-type Trpase). Thus, Tyr-74 and His-463 are important for the substrate specificity of Trpase for L-Trp. H463F Trpase is not inhibited by a potent inhibitor of wild-type Trpase, oxindolyl-L-alanine, and does not exhibit the pK(a) of 6.0 seen in previous pH dependence studies [Kiick, D. M., and Phillips, R. S. (1988) Biochemistry 27, 7333]. These results suggest that His-463 may be the catalytic base with a pK(a) of 6.0 and Tyr-74 may be a general acid catalyst for the elimination step, as we found previously with tyrosine phenol-lyase [Chen, H., Demidkina, T. V., and Phillips, R. S. (1995) Biochemistry 34, 12776]. H463F Trpase reacts with L-Trp and S-ethyl-L-cysteine in rapid-scanning stopped-flow experiments to form equilibrating mixtures of external aldimine and quinonoid intermediates, similar to those observed with wild-type Trpase. In contrast to the results with wild-type Trpase, the addition of benzimidazole to reactions of H463F Trpase with L-Trp does not result in the formation of an aminoacrylate intermediate. However, addition of benzimidazole with S-ethyl-L-cysteine results in the formation of an aminoacrylate intermediate, with lambda(max) at 345 nm, as was seen previously with wild-type Trpase [Phillips, R. S. (1991) Biochemistry 30, 5927]. This suggests that His-463 plays a specific role in the elimination step of the reaction of L-Trp. Refolding of equimolar mixtures of H463F and Y74F Trpase after unfolding in 4 M guanidine hydrochloride results in a dramatic increase in activity with L-Trp, indicating the formation of a hybrid H463F/Y74F dimer with one normal active site.

Tryptophan indole-lyase from Proteus vulgaris: kinetic and spectral properties.

An efficient method for purification of recombinant tryptophanase from Proteus vulgaris was developed. Catalytic properties of the enzyme in reactions with L-tryptophan and some other substrates as well as competitive inhibition by various amino acids in the reaction with S-o-nitrophenyl-L-cysteine were studied. Absorption and circular dichroism spectra of holotryptophanase and its complexes with characteristic inhibitors modeling the structure of the principal reaction intermediates were examined. Kinetic and spectral properties of two tryptophanases which markedly differ in their primary structures are compared. It was found that although the spectral properties of the holoenzymes and their complexes with amino acid inhibitors are different, the principal kinetic properties of the enzymes from Proteus vulgaris and Escherichia coli are analogous. This indicates structural similarity of their active sites.

Proton Transfer and Carbon−Carbon Bond Cleavage in the Elimination of Indole Catalyzed by Escherichia coli Tryptophan Indole-Lyase

Tryptophan indole-lyase from Escherichia coli catalyzes the reversible cleavage of l-tryptophan to indole and ammonium pyruvate. This reaction is mechanistically interesting since it involves the elimination of an aromatic carbon leaving group. We have been studying the mechanism of tryptophan indole-lyase using rapid-scanning stopped-flow spectrophotometry. Recently, we demonstrated that the rate constant for α-aminoacrylate intermediate formation from α-2H-l-tryptophan exhibits an isotope effect of 3.0 (Sloan, M. J.; Phillips, R. S. Biochemistry 1996, 35, 16165−16173). We have confirmed this previous result (Dk = 2.99 ± 0.30) and we have now found that β,β-di-2H-l-tryptophan also exhibits a secondary isotope effect (Dk = 1.17 ± 0.03) on the elimination reaction. Furthermore, α,β,β-tri-2H-l-tryptophan exhibits a multiple isotope effect (Dk = 4.42 ± 0.67) on the elimination of indole. In addition, there is a significant solvent isotope effect (Dk = 1.79 ± 0.11) on indole elimination in D2O. This solvent isotope effect combines with the effect of α-deuterium, since elimination of α-2H-l-tryptophan in D2O exhibits Dk = 4.30 ± 0.16. In addition, the rate constant for indole elimination shows a linear Eyring plot between 5 and 35 °C. In the direction of tryptophan synthesis, the reaction of the α-aminoacrylate intermediate with indole to form a quinonoid intermediate also exhibits a kinetic isotope effect for 3-2H-indole, with Dk = 1.88 ± 0.19. In contrast to our expectations, the results suggest that the proton transfer and carbon−carbon bond cleavage in the elimination reaction are very nearly simultaneous and that the indolenine structure is a transient intermediate which occupies a very shallow well on the reaction coordinate, or a transition state, in the reaction of Trpase.

Cleavage of Escherichia coli tryptophan indole-lyase by trypsin at Lys406 affects the transmission of conformational changes associated with monovalent cation activation.

Escherichia coli tryptophan indole-lyase (Trpase) is a pyridoxal 5'-phosphate(pyridoxal-P)-dependent enzyme which catalyzes the hydrolytic cleavage of L-tryptophan to indole and ammonium pyruvate. This enzyme is strongly activated by K+ and similar monovalent cations, and the spectrum of the pyridoxal-P cofactor is also affected by pH and cations. Treatment of Trpase with trypsin results in a 20-100-fold decrease in elimination activity, depending on the substrate, concomitant with a change in the relative amounts of the 337 nm and 420 nm forms of the bound pyridoxal-P, and a shift in the lambda(max) from 420 nm to 423 nm. In addition, the pH sensitivity of the pyridoxal-P cofactor is eliminated after trypsin treatment. Nicked Trpase exhibits only fourfold activation by K+, compared with about 50-fold for native enzyme, but the K(A) for K+ is unaffected. Both the native and trypsin-nicked Trpase react with amino acids to form equilibrating mixtures of external aldimine and quinonoid intermediates in rapid-scanning stopped-flow experiments. However, the rate constant for quinonoid intermediate formation from L-tryptophan is reduced by at least 400-fold by treatment with trypsin. In contrast, the rate constant for formation of quinonoid intermediates of L-alanine and S-ethyl-L-cysteine is affected only twofold or less by trypsin treatment. The site of trypsin cleavage was identified by electrospray-ionization mass spectrometry as Lys406, which is predicted to lie on a flexible surface loop. Some active-site residues, particularly Arg419, which is predicted by sequence similarity to be the substrate alpha-carboxylate-binding site, and His463, are located in the sequence between Lys406 and the C-terminus. Hence, cleavage of the peptide bond of E. coli Trpase at Lys406 probably affects the change from active to inactive conformations that normally takes place in the presence of activating monovalent cations.

Effects of tyrosine ring fluorination on rates and equilibria of formation of intermediates in the reactions of carbon-carbon lyases.

The interactions of ring fluorinated analogs of tyrosine with tyrosine phenol-lyase and tryptophan indole-lyase (tryptophanase) were studied by rapid-scanning stopped-flow spectrophotometry. The reaction of L-tyrosine with tyrosine phenol-lyase resulted in rapid formation of a small absorbance peak at 500 nm, attributed to a quinonoid intermediate. The reaction of 3-fluoro-L-tyrosine with tyrosine phenol-lyase resulted in a peak at 500 nm with much higher absorbance, as did the reaction of 3,5-difluoro-L-tyrosine, due to increased accumulation of quinonoid intermediates. In constrast, complexes with 2-fluoro-L-tyrosine, 2,3-difluoro-L-tyrosine, 2,5-difluoro-L-tyrosine, and 2,6-difluoro-L-tyrosine exhibited much lower absorbance intensity at 500 nm. The rate constant for quinonoid intermediate formation from 3-fluoro-L-tyrosine was comparable to that for L-tyrosine. However, 3,5-difluoro-L-tyrosine reacted to form a quinonoid intermediate at about half the rate of L-tyrosine, while 2,3-difluoro-L-tyrosine reacted at twice the rate of L-tyrosine. In addition, the 2-substituted difluorotyrosines exhibited an intermediate, which was formed rapidly, absorbing strongly at about 340 nm, which is likely due to a gem-diamine intermediate. Tyrosine is not a substrate for tryptophan indole-lyase; the reaction of tryptophan indole-lyase with L-tyrosine resulted in formation of external aldimine, which absorbed at 420 nm, and a very small absorbance peak at 500 nm. 3-Fluoro-L-tyrosine reacted with tryptophan indole-lyase to produce a prominent quinonoid absorbance peak at 500 nm, whereas L-tyrosine, 2-fluoro-L-tyrosine, and all difluoro-L-tyrosines, had a much reduced intensity for this peak. Thus, the presence of ring fluorine substituents in L-tyrosine that are remote from the site of the chemical transformation has significant effects on the rates and equilibria of intermediate formation in the reactions with both tyrosine phenol-lyase and tryptophan indole-lyase. Although it is commonly thought that fluorine substitution will not result in any significant steric effects, our results suggest that the effects of fluorine substitution in the reactions of fluorinated tyrosines with tyrosine phenol-lyase and tryptophan indole-lyase are due to a combination of steric and electronic effects.

Effects of alpha-deuteration and of aza and thia analogs of L-tryptophan on formation of intermediates in the reaction of Escherichia coli tryptophan indole-lyase.

Tryptophan indole-lyase catalyzes the hydrolytic cleavage of L-tryptophan to indole and ammonium pyruvate. After the enzyme is mixed with L-tryptophan in the rapid-scanning stopped-flow spectrophotometer, there is an absorbance increase at 505 nm in the pre-steady state attributed to formation of a quinonoid intermediate, which occurs in at least three consecutive first-order phases. Reaction with [alpha-2H]-L-tryptophan results in significant primary kinetic isotope effects on the first two phases, and there is a significant isotope effect on the amplitude of the absorbance increase in the second phase. This result suggests that proton transfer to carbon to form the indolenine intermediate is relatively slow and is probably at least partially rate-determining. Reaction of L-tryptophan in the presence of benzimidazole results in a rapid increase in absorbance in the first phase, followed by a decrease in absorbance in the second phase, with rate constants very similar to those observed without benzimidazole. We have also examined aza and thia analogs of L-tryptophan, with the benzene ring of the indole replaced by pyridine or thiophene. Both 4,5-thiatryptophan and 6,7-thiatryptophan form quinonoid intermediates in the reaction with tryptophan indole-lyase; however, 6,7-thiatryptophan is a better substrate (kcat/K(m) = 32% of L-trp) for tryptophan indole-lyase than is 4,5-thiatryptophan (kcat/K(m) = 4% of L-trp). Benzimidazole affects the pre-steady-state reaction of 6,7-thiatryptophan in a way similar to L-tryptophan, while benzimidazole does not affect the pre-steady-state reaction of 4,5-thiatryptophan. 4-Aza-, 5-aza-, 6-aza-, and 7-aza-L-tryptophan are all very slow substrates (kcat < 1% of L-trp) for Escherichia coli tryptophan indole-lyase. beta-Indazolyl-L-alanine is a relatively good substrate and exhibits a quinonoid intermediate in its reaction with tryptophan indole-lyase. 6-Aza- and 7-azatryptophan accumulate quinonoid intermediates in the reaction with tryptophan indole-lyase, whereas 4-aza- and 5-azatryptophans do not significantly accumulate quinonoid intermediates, and these latter compounds exhibit very high K(m) values. Addition of benzimidazole does not change the rapid-scanning stopped-flow spectra of 6-aza- and 7-azatryptophan. This suggests that the rate-determining step in the reaction changes depending on the position and type of heteroatom substitution. For 6-aza- and 7-azatryptophan, the very slow rates of elimination may be due to slow C-protonation of the azaindole, while for 4,5-thiatryptophan, the elimination of thienopyrrole is probably slow. Of all analogs examined, 6,7-thiatryptophan is most similar to tryptophan in its reaction with E. coli tryptophan indole-lyase.

Site-Directed Mutagenesis of His343Ala in Citrobacter freundii Tyrosine Phenol-Lyase. Effects on the Kinetic Mechanism and Rate-Determining Step

His343 in Citrobacter freundii tyrosine phenol-lyase is conserved in all known sequences of both tyrosine phenol-lyase and tryptophan indole-lyase; it is located near the active-site Lys257 in C. freundii tyrosine phenol-lyase [Antson, A. A., Demidkina, T. V., Gollnick, P., Danter, Z., Von Tersch, R. L., Long, J., Berezhnoy, S. N., Phillips, R. S., Harutyunyan, E. H. & Wilson, K. S. (1993) Biochemistry 32, 4195–4206]. In order to evaluate the role of His343 in the reaction mechanism of tyrosine phenol-lyase and tryptophan indolelyase, we have mutated it to Ala; the former mutant is referred to as [H343A]tyrosine phenol lyase. All substrates for α,/β-elimination (except S -ethyl-l-cysteine) exhibited lower kcat (10–30%) and kcat/Km (1–10%) values with [H343A]tyrosine phenol-lyase than with the wild-type enzyme. The mutant also shows slower rates of deuterium isotope exchange for l-phenylalanine and l-methionine than does the wild type. The pH-dependent behavior in the reaction of 3-fluoro-l-tyrosine with wild-type tyrosine phenol-lyase is identical to that of l-tyrosine described previously [Kiick, D. M. & Phillips, R. S. (1988) Biochemistry 27, 7333–7338]. The pH profile of kcatKm for this reaction exhibits two pKa values with an average of 7.7 ± 0.2, indicating that the catalytic mechanism requires two essential basic groups. The pH profile of kcat/Km for 3-fluoro-L-tyrosine with [H343A]tyrosine phenol-lyase also exhibits two pKa values with an average of 7.8 ± 0.3. However, kcat for 3-fluoro-l-tyrosine is pH-dependent for the mutant, exhibiting two pKa values with an average of about 7.8, whereas it is pH-independent for the wild type. Steady-state kinetic isotope effects on the reactions with wild-type and [H343A]tyrosine phenol-lyase were examined at various pH values. For the wild type, the values of the isotope effects on fccat and kcat/Km for 3-fluoro-l-[α-2H]-tyrosine are independent of pH and equal to 3.9 ± 0.2 and 2.2±0.3, respectively, while the corresponding values for [H343A]tyrosine phenol-lyase are 5.4 ± 0.2 and 3.8 ± 0.3, respectively. Based on these observations and the pH data, we conclude that α-proton abstraction is apparently the rate-determining step in the reaction of 3-fluoro-l-tyrosine with [H343A]tyrosine phenol-lyase; however, quinonoid intermediate formation (140 s −1) is much faster than kcat of 3-fluoro-L-tyrosine (1.4 s −1) for wild-type tyrosine phenol-lyase; thus, the mutation of His343Ala has apparently changed the rate-determining step of the reaction with 3-fluoro-l-tyrosine. l-Methionine, l-homoserine, l-phenylalanine and l-alanine form quinonoid intermediates with wild-type tyrosine phenol-lyase which exhibit absorbance peaks at about 500 nm but the intensity of these absorbance peaks is dramatically reduced for the mutant. Single-wavelength stopped-flow kinetic measurements demonstrate that [H343A]tyrosine phenol-lyase exhibits slower rate constants for α-deprotonation and faster rate constants for reprotonation than does the wild-type. Taken together, these results demonstrate that, while His343 is not an essential basic group in tyrosine phenol-lyase, it does play an important function in catalysis, possibly by facilitating the conformational change from an ‘open’ to ‘closed’ form when substrates bind.

Site-Directed Mutagenesis of His343Ala in Citrobacter freundii Tyrosine Phenol-Lyase. Effects on the Kinetic Mechanism and Rate-Determining Step

His343 in Citrobacter freundii tyrosine phenol-lyase is conserved in all known sequences of both tyrosine phenol-lyase and tryptophan indole-lyase; it is located near the active-site Lys257 in C. freundii tyrosine phenol-lyase [Antson, A. A., Demidkina, T. V., Gollnick, P., Danter, Z., Von Tersch, R.L., Long, J., Berezhnoy, S. N., Phillips, R. S., Harutyunyan, E. H. & Wilson, K. S. (1993) Biochemistry 32, 4195--4206]. In order to evaluate the role of His343 in the reaction mechanism of tyrosine phenol-lyase and tryptophan indole-lyase, we have mutated it to Ala; the former mutant is referred to as [H343A]tyrosine phenol lyase. All substrates for alpha, beta-elimination (except S-ethyl-L-cysteine) exhibited lower kcat (10-30%) and kcat/Km (1-10%) values with [H343A]tyrosine phenol-lyase than with the wild-type enzyme. The mutant also shows slower rates of deuterium isotope exchange for L-phenylalanine and L-methionine than does the wild type. The pH-dependent behavior in the reaction of 3-fluoro-L-tyrosine with wild-type tyrosine phenol-lyase is identical to that of L-tyrosine described previously [Kiick, D. M. & Phillips, R. S. (1988) Biochemistry 27, 7333-7338]. The pH profile of kcat/Km for this reaction exhibits two pKa values with an average of 7.7 +/- 0.2, indicating that the catalytic mechanism requires two essential basic groups. The pH profile of kcat/Km for 3-fluoro-L-tyrosine with [H343A]tyrosine phenol-lyase also exhibits two pKa values with an average of 7.8 +/- 0.3. However, kcat for 3-fluoro-L-tyrosine is pH-dependent for the mutant, exhibiting two pKa values with an average of about 7.8, whereas it is pH-independent for the wild type. Steady-state kinetic isotope effects on the reactions with wild-type and [H343A]tyrosine phenol-lyase were examined at various pH values. For the wild type, the values of the isotope effects on kcat and kcat/Km for 3-fluoro-L-[alpha-2H]-tyrosine are independent of pH and equal to 3.9 +/- 0.2 and 2.2 +/- 0.3, respectively, while the corresponding values for [H343A]tyrosine phenol-lyase are 5.4 +/- 0.2 and 3.8 +/- 0.3, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

The Folding Characteristics of Tryptophanase from Escherichia coli

The unfolding and refolding characteristics of Escherichia coli tryptophanase (tryptophan indole-lyase) [EC 4.1.99.1] in guanidine hydrochloride were studied. Tryptophanase unfolded by first dissociating its coenzyme, pyridoxal 5'-phosphate, from the active site. This dissociation caused a significant destabilization of structure, and global unfolding of the protein followed. During this global unfolding step, an intermediate was formed which had a strong tendency to aggregate irreversibly, as detected by light scattering experiments. Tryptophanase was unable to refold quantitatively after unfolding in 4 M guanidine hydrochloride. The low refolding yield was due to non-specific aggregation which occurs during refolding. Various conditions which limited this aggregation were probed, and it was found that by initiating the refolding reaction at low temperature, the aggregation of tryptophanase folding intermediates during the reaction could be avoided to a certain extent, and the refolding yield improved.

The mechanism of Escherichia coli tryptophan indole-lyase: substituent effects on steady-state and pre-steady-state kinetic parameters for aryl-substituted tryptophan derivatives

We have examined the reaction of Escherichia coli tryptophan indole-lyase with fluoro, chloro, methyl and hydroxytryptophans using steady-state kinetics, rapid-scanning and single wavelength stopped-flow spectrophotometry, and rapid chemical quench methods. All of the 16 tryptophan derivatives examined are substrates for α,β-elimination catalyzed by tryptophan indole-lyase. The steady-state kinetic parameter, k cat/ K m, did not show a consistent trend with the steric bulk of the substituent, but K m increased for larger substituents. Rapid-scanning stopped-flow spectra show that all tryptophan analogues undergo covalent reaction with the pyridoxal-5′-phosphate cofactor to give equilibrating mixtures of external aldimine and quinonoid intermediates, but the relative amounts of each intermediate are strongly dependent on the nature and position of the substituent. The dissociation constants for external aldimine formation, K d, obtained from single-wavelength stopped-flow experiments decreased for most substituted tryptophans, which suggests that part of the binding energy is derived from hydrophobic interactions between the enzyme and the indole ring of tryptophan. In contrast, the rate constants of quinonoid intermediate formation and reprotonation and of indole elimination were quite variable, depending on the position and the nature of the substituent. Overall, 6-substituted tryptophans have the most consistent reactivity, which indicates that there may be space in the enzyme active site near the 6-position. There is a good linear correlation between log ( k cat/ K m) and log ( k f/ K d) (apparent second order rate constant for quinonoid intermediate formation), with a slope of 0.66. This suggests that quinonoid intermediate formation contributes only about 66% of the activation energy for the reaction, and thus a later step in the reaction must be partially rate-limiting. Rapid chemical quench experiments demonstrate a ‘burst’ of indole in the reaction of l-tryptophan under single turnover conditions, confirming that a step subsequent to the elimination is partially rate-determining. In contrast, 5-methyl- l-tryptophan does not exhibit a significant ‘burst’, suggesting that 5-methylindole elimination is nearly completely rate-determining. These results support the proposed mechanism and demonstrate that there are significant effects of aryl substituents on the distribution of covalent intermediates and on the rate-determining step in the α,β-elimination reaction catalyzed by E. coli tryptophan indole-lyase.