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We have designed, synthesized and evaluated homotryptophan analogues as possible mechanism-based inhibitors for Escherichia coli tryptophan indole-lyase (tryptophanase, TIL, E.C. 4.1.99.1). As a quinonoid structure is an intermediate in the reaction mechanism of TIL, we anticipated that homologation of the physiological substrate, L-Trp would provide analogues resembling the transition state for β-elimination, and potentially inhibit TIL. Our results demonstrate that L-homotryptophan (1a) is a moderate competitive inhibitor of TIL, with Ki=67 μM, whereas L-bishomotryptophan (1b) displays more potent inhibition, with Ki=4.7 μM. Pre-steady-state kinetics indicated the formation of an external aldimine and quinonoid with 1a, but only the formation of an external aldimine for 1b, suggesting differences in the inhibition mechanism. These results demonstrate that formation of a quinonoid complex is not required for strong inhibition. In addition, the Trp analogues were evaluated as inhibitors of Salmonella typhimurium Trp synthase. Our results indicate that compound 1b is at least 25-fold more selective toward TIL than Trp synthase. We report that compound 1b is comparable to the most potent inhibitor previously reported, while displaying high selectivity for TIL. Thus, 1b is a potential lead for the development of novel antibacterials., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Oligomeric enzymes can undergo a reversible loss of activity at low temperatures. One such enzyme is tryptophanase (Trpase) from Escherichia coli. Trpase is a pyridoxal phosphate (PLP)-dependent tetrameric enzyme with a Mw of 210 kD. PLP is covalently bound through an enamine bond to Lys270 at the active site. The incubation of holo E. coli Trpases at 2 degrees C for 20 h results in breaking this enamine bond and PLP release, as well as a reversible loss of activity and dissociation into dimers. This sequence of events is termed cold lability and its understanding bears relevance to protein stability and shelf life., Results: We studied the reversible cold lability of E. coli Trpase and its Y74F, C298S and W330F mutants. In contrast to the holo E. coli Trpase all apo forms of Trpase dissociated into dimers already at 25 degrees C and even further upon cooling to 2 degrees C. The crystal structures of the two mutants, Y74F and C298S in their apo form were determined at 1.9A resolution. These apo mutants were found in an open conformation compared to the closed conformation found for P. vulgaris in its holo form. This conformational change is further supported by a high pressure study., Conclusion: We suggest that cold lability of E. coli Trpases is primarily affected by PLP release. The enhanced loss of activity of the three mutants is presumably due to the reduced size of the side chain of the amino acids. This prevents the tight assembly of the active tetramer, making it more susceptible to the cold driven changes in hydrophobic interactions which facilitate PLP release. The hydrophobic interactions along the non catalytic interface overshadow the effect of point mutations and may account for the differences in the dissociation of E. coli Trpase to dimers and P. vulgaris Trpase to monomers.

Bacteria undergo significant changes during adherence to surfaces and biofilm development. Cell-to-cell signalling molecules are known to be involved in these phenotypic adaptations to the sessile mode of life. We demonstrated previously that indole can act as an extracellular signal to regulate biofilm formation in E. coli. To identify proteins over- or under-expressed in response to E. coli biofilm formation and indole signalling, we compared the proteomes of the E. coli S17-1 wild-type and 3714 (S17-1 tnaA::Tn5) tryptophanase-negative mutant cells (which don't produce indole) grown as suspensions or biofilms in the presence or absence of exogenous indole. From computer-assisted image analysis, 407 spots were discriminated on two-dimensional electropherograms. Principal component analysis (PCA) of the electropherograms did not discriminate between the proteomes of the wild-type and mutant cells grown as suspensions indicating that indole has a limited impact onto protein expression of planktonic cells. The first principal component extracted by PCA, after standardization of the observations, opposed planktonic and biofilm cells confirming the existence of changes in protein expression during E. coli biofilm formation. Among proteins over- or under-expressed by both sessile wild-type and mutant cells, we identified metabolic enzymes, transporters, proteins involved in the translation and transcription machinery, stress response and regulation, and signalling proteins. The wild-type and mutant strains grown as biofilms in the presence of indole were discriminated by the second component. The role of some proteins whose expression was altered in biofilm bacteria compared to suspended counterparts is discussed.

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.

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.

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.

The mechanism of pyridoxal phosphate (PLP) binding to apotryptophanase was investigated using stopped-flow kinetics with wild type (WT), W330F and C298S mutants. Based on the dependence of the rate constants on PLP concentrations for the fast and slow phases detected, two mechanistic schemes were proposed. For the WT and C298S mutant, the slow process is due to an isomerization of the aldimine complex after its formation, and not to the binding to an alternative conformation of the apoenzyme, which is the case proposed for the W330F mutant. It is suggested that during the cold inactivation process a conformational change precedes the aldimine bond cleavage. For the W330F apotryptophanase, another conformational change occurs subsequent to the aldimine bond cleavage.

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.

The kinetics and mechanism of reversible cold inactivation of the tetrameric enzyme tryptophanase have been studied. Cold inactivation is shown to occur slowly in the presence of K+ ions and much faster in their absence. The W330F mutant tryptophanase undergoes rapid cold inactivation even in the presence of K+ ions. In all cases the inactivation is accompanied by a decrease of the coenzyme 420-nm CD and absorption peaks and a shift of the latter peak to shorter wavelengths. The spectral changes and the NaBH4 test indicate that cooling of tryptophanase leads to breaking of the internal aldimine bond and release of the coenzyme. HPLC analysis showed that the ensuing apoenzyme dissociates into dimers. The dissociation depends on the nature and concentration of anions in the buffer solution. It readily occurs at low protein concentrations in the presence of salting-in anions Cl-, NO3- and I-, whereas salting-out anions, especially HPO4(2-), hinder the dissociation. K+ ions do not influence the dissociation of the apoenzyme, but partially protect holotryptophanase from cold inactivation. Thus, the two processes, cold inactivation of tryptophanase and dissociation of its apoform into dimers exhibit different dependencies on K+ ions and anions.

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.

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.

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 alpha, beta-elimination catalyzed by tryptophan indole-lyase. The steady-state kinetic parameter, kcat/Km, did not show a consistent trend with the steric bulk of the substituent, but Km 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, Kd, 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 (kcat/Km) and log (kf/Kd) (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 alpha, beta-elimination reaction catalyzed by E. coli tryptophan indole-lyase.

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.

Lysine 269 in Escherichia coli tryptophan indole-lyase (tryptophanase) has been changed to arginine by site-directed mutagenesis. The resultant K269R mutant enzyme exhibits kcat values about 10% those of the wild-type enzyme with S-(o-nitrophenyl)-L-cysteine, L-tryptophan, and S-benzyl-L-cysteine, while kcat/Km values are reduced to 2% or less. The pH profile of kcat/Km for S-benzyl-L-cysteine for the mutant enzyme exhibits two pK alpha values which are too close to separate, with an average value of 7.6, while the wild-type enzyme exhibits pK alpha values of 6.0 and 7.8. The pK alpha for the interconversion of the 335 and 412 nm forms of the K269R enzyme is 8.3, while the wild-type enzyme exhibits a pK alpha of 7.4. Steady-state kinetic isotope effects on the reaction of [alpha-2H]S-benzyl-L-cysteine with the K269R mutant enzyme (Dkcat = 2.0; D(kcat/Km) = 3.9) are larger than those of the wild-type enzyme (Dkcat = 1.4; D(kcat/Km) = 2.9). Rapid scanning stopped-flow kinetic studies demonstrate that the K269R mutant enzyme does not accumulate quinonoid intermediates with L-alanine, L-tryptophan, or S-methyl-L-cysteine, but does form quinonoid absorption peaks in complexes with S-benzyl-L-cysteine and oxidolyl-L-alanine, whereas wild-type enzyme forms prominent quinonoid bands with all these amino acids. Single wavelength stopped-flow kinetic studies demonstrate that the alpha-deprotonation of S-benzyl-L-cysteine is 6-fold slower in the K269R mutant enzyme, while the intrinsic deuterium kinetic isotope effect is less for the K269R enzyme (Dk = 4.2) than for the wild-type (Dk = 7.9). The decay of the K269R quinonoid intermediate in the presence of benzimidazole is 7.1-fold slower than that of the wild-type enzyme. These results demonstrate that Lys-269 plays a significant role in the conformational changes or electrostatic effects obligatory to the formation and decomposition of the quinonoid intermediate, although it is not an essential basic residue.

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.

The two tryptophan residues, Trp-248 and Trp-330, in tryptophan indole-lyase (tryptophanase) from E. coli have been separately mutated to phenylalanine using site-directed mutagenesis. Both single tryptophan mutant enzymes have full catalytic activity, but exhibit different fluorescence and near-UV circular dichroism spectra. These results indicate that Trp-330 is more deeply buried than is Trp-248, and is in a more asymmetric environment. Neither residue reacts with N-bromosuccinimide (NBS), although tryptophan indole-lyase is inactivated by NBS. These results demonstrate that the tryptophan residues in tryptophan indole-lyase are not catalytically essential.

Escherichia coli tryptophan indole-lyase (tryptophanase) mutants, with cysteine residues 294 and 298 selectively replaced by serines, have been prepared by site-directed mutagenesis. Both mutant enzymes are highly active for beta-elimination reactions measured with both L-tryptophan and S-(o-nitrophenyl)-L-cysteine. The Cys-294----Ser mutant enzyme is virtually identical to the wild type with respect to pyridoxal phosphate binding (KCO = 2 microM), cofactor absorption spectrum (lambda max = 420 and 337 nm) and pH dependence (pK alpha = 7.3), pH profile for catalysis, and rate of bromopyruvic acid inactivation. In contrast, the Cys-298----Ser mutant enzyme exhibits a reduced affinity for pyridoxal phosphate (KCO = 6 microM), a shift in the cofactor absorption spectrum to 414 nm and an altered pK alpha = 8.5, an alkaline shift in the pH profile for catalysis, and resistance to inactivation of the apoenzyme by bromopyruvic acid. The C298S mutant enzyme (wherein cysteine 298 is altered to serine) also undergoes an isomerization to an unreactive state upon storage at 4 degrees C. These results demonstrate that the sulfhydryl groups of Cys-294 and Cys-298 are catalytically nonessential. However, these data suggest that Cys-298 is located within or very near the active site of the enzyme and is the reactive cysteine residue previously observed by others.

Analysis of the pH dependence of the kinetic parameters and competitive inhibitor Ki values for tryptophan indole-lyase suggests two enzymic groups must be unprotonated in order to facilitate binding and catalysis of tryptophan. The V/K for tryptophan and the pKi for oxindolyl-L-alanine, a putative transition state analogue and competitive inhibitor, decrease below two pK values of 7.6 and 6.0, while the Ki for L-alanine, also a competitive inhibitor, is 3300-fold larger (20 mM) than that for oxindolyl-L-alanine and increases below a single pK of 7.6. A single pK of 7.6 is also observed in the V/K profile for the alternate substrate, S-methyl-L-cysteine. Therefore, the enzymic group with a pK of 7.6 is responsible for proton abstraction at the 2-position of tryptophan, while the enzymic group with a pK of 6.0 interacts with the indole portion of tryptophan and probably catalyzes formation of the indolenine tautomer of tryptophan (in concert with proton transfer to C-3 of indole from the group with pK 7.6) to facilitate carbon-carbon bond cleavage and elimination of indole. The pH variation of the primary deuterium isotope effects for proton abstraction at the 2-position of tryptophan (DV = 2.5 and D(V/Ktrp) = 2.8) are pH independent, while the Vmax for tryptophan or S-methyl-L-cysteine is the same and also pH independent. Thus, substrates bind only to the correctly protonated form of the enzyme. Further, tryptophan is not sticky, and the pK values observed in both V/K profiles are the correct ones.(ABSTRACT TRUNCATED AT 250 WORDS)