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

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

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

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

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

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

A simple colorimetric method for the determination of tryptophan

A simple, sensitive, and reproducible colorimetric method for the determination of tryptophan in amounts as low as 2 μg is described. It is based on the oxidation of tryptophan by sodium nitrite and the coupling of the oxidized product to the leucodye N-1-(naphthyl)ethylenediamine dihydrochloride. The purple-pink product has an absorption maximum at 550 nm. There is no interference by carbohydrates, other amino acids, neutral salts, or a number of other compounds likely to be found in tissue hydrolysates. A number of indole derivatives including indole-3-acetic acid also react to give a colored product. Dipeptides containing tryptophan are much less reactive than free tryptophan; hence proteins must be hydrolyzed completely for the method to be useful. The assay is carried out at room temperature and can be modified easily to increase or decrease its sensitivity. It has been employed to determine the tryptophan content of a number of proteins following alkaline hydrolysis. Generally, values obtained were in close agreement with values reported in the literature.     

Photomonomerization of pyrimidine dimers by indoles and proteins.

Model systems for the study of photoreactivation have been developed that utilize a variety of indole derivatives. These systems can split uracil cis-syn cyclobutadipyrimidine, either free or in RNA, when irradiated at wave-lengths absorbed only by the indole moiety. The ability of indole compounds to split dimers is closely related to their electronic properties. Those of high electron-donor capacity such as indole, 3-methylindole, indole-3-acetic acid, 5-hydroxytryptophan and tryptophan are good photosensitizers, with efficacy in that order. Indoles with electron-withdrawing substituents such as indole-3-carboxylic acid, indole-3-aldehyde and oxindole are inactive in the monomerization reaction. These findings support the proposed mechanism that the photosensitized monomerization occurs as a result of electron transfer from the excited indole molecules to the pyrimidine bases.Proteins containing fully exposed tryptophan residues (chicken egg white lysozyme and bovine diisopropylphosphoryltrypsin) also cause the splitting of the ¹⁴C-labeled dimers under the same conditions. In the case of lysozyme the quantum yield of monomerization is similar to that of free tryptophan. Much of the monomerization ability of lysozyme was lost after the solvent-available tryptophan had been oxidized by treatment with N-bromosuccinimide. Bovine pancreatic ribonuclease A, a protein devoid of tryptophan, failed to exhibit photosensitized monomerization of uracil dimers. The biological implication of these reactions involving a protein with an exposed tryptophan residue is discussed.Although indoles are able to split the dimers in RNA, they fail to photo-reactivate u.v.-damaged TMV-RNA. Indole-3-acetic acid, 3-methylindole and 5-hydroxytryptophan rapidly inactivate viral RNA when irradiated at 313 nm, possibly because of side reactions.     

The reactivity of tryptophan residues in proteins. Stopped-flow kinetics of fluorescence quenching.

The quenching of tryptophan fluorescence by N-bromosuccinamide, studied by the fluorescence stopped-flow technique, was used to compare the reactivities of tryptophan residues in protein molecules. The reaction of N-bromosuccinamide with the indole group of N-acetyltryptophanamide, a model compound for bound tryptophan, followed second-order kinetics with a rate constant of (7.8 +/- 0.8) . 10(5) dm3 . mol-1 . s-1 at 23 degrees C. The rate does not depend on ionic strength or on the pH near neutrality. The non-fluorescent intermediate formed from N-acetyltryptophanamide on the reaction with N-bromosuccinamide appears to be a bromohydrin compound. The second-order rate constant for fluorescence quenching of tryptophan in Gly-Trp-Gly by N-bromosuccinamide was very similar, (8.8 +/- 0.8) . 10(5) dm3 . mol-1 . s-1. Apocytochrome c has the conformation of a random coil with the single tryptophan largely exposed to the solvent. The rate constant for the fluorescence quenching of the tryptophan in apocytochrome c by N-bromosuccinamide was (3.7 +/- 0.3) . 10(5) dm3 . mol-1 . s-1. The fluorescence quenching by N-bromosuccinamide of the tryptophan residues incorporated in alpha-chymotrypsin at pH 7.0 showed three exponential terms from which the following rate constants were derived: 1.74 . 10(5), 0.56 . 10(5) and 0.11 . 10(5) dm3 . mol-1 . s-1. This protein is known to have eight tryptophan residues in the native state, six residues at the surface, and two buried. Three of the surface tryptophans have the indole rings protruding out of the molecule and may account for the fastest kinetic phase of the quenching process. The intermediate phase may be due to three surface tryptophans whose indole rings point inwards, and the slowest to the two interior tryptophan residues.     

A specific method for determination of free tryptophan and endogenous tryptophan in Escherichia coli.

A sensitive, simple spectrofluorometric technique for determination of tryptophan inamounts as small as 10 pmol is described. It is based on tryptophanase hydrolysis of tryptophan and spectrofluorometric analysis of the resulting indole. The relationship between released indole and fluorescence is linear over three orders of magnitude. The method is free from interference by other amino acids, polar indole derivatives, and a number of other compounds found in cell extracts or used in bacterial growth media. The method is rapid, reproducible, and accurate. A simple method for extraction and measurement of endogenous free tryptophan from bacterial cells is also described.     

Serine modulates substrate channeling in tryptophan synthase. A novel intersubunit triggering mechanism

Tryptophan synthase, an alpha 2 beta 2 complex, is a classic example of an enzyme that is thought to "channel" a metabolic intermediate (indole) from the active site of the alpha subunit to the active site of the beta subunit. We now examine the kinetics of substrate channeling by tryptophan synthase directly by chemical quench-flow and stopped-flow methods. The conversion of indole-3-glycerol phosphate (IGP) to tryptophan at the active site proceeds at a rate of 24 s-1, which is limited by the rate of cleavage of IGP to produce indole (alpha reaction). In a single turnover experiment monitoring the conversion of radiolabeled IGP to tryptophan, only a trace of indole is detectable (less than or equal to 1% of the IGP), implying that the reaction of indole to form tryptophan must be quite fast (greater than or equal to 1000 s-1). The rate of reaction of indole from solution is much too slow (40 s-1 under identical conditions) to account for the negligible accumulation of indole in a single turnover. Therefore, the indole produced at the alpha site must be rapidly channeled to the beta site, where it reacts with serine to form tryptophan: channeling and the reaction of indole to form tryptophan must each occur at rates greater than or equal to 1000 s-1. Steady-state turnover is limited by the slow rate of tryptophan release (8 s-1). In the absence of serine, the cleavage of IGP to indole is limited by a change in protein conformation at a rate of 0.16 s-1. When the alpha beta reaction is initiated by mixing enzyme with IGP and serine simultaneously, there is a lag in the cleavage IGP and formation of tryptophan. The kinetics of the lag correspond to the rate of formation of the aminoacrylate in the reaction of serine with pyridoxal phosphate at the beta site, measured by stopped-flow methods (45 s-1). There is also a change in protein fluorescence, suggestive of a change in protein conformation, occurring at the same rate. Substitution of cysteine for serine leads to a longer lag in the kinetics of IGP cleavage and a correspondingly slower rate of formation of the aminoacrylate (6 s-1). Thus, the reaction of serine at the beta site modulates the alpha reaction such that the formation of the aminoacrylate leads to a change in protein conformation that is transmitted to the alpha site to enhance the rate of IGP cleavage 150-fold.(ABSTRACT TRUNCATED AT 400 WORDS)     

The modification of the lone tryptophan residue in human serum albumin by 2-hydroxy-5-nitrobenzyl bromide. Characterization of the modified protein and the binding of L-tryptophan and benzodiazepines to the tryptophan-modified albumin.

The possible function of the lone tryptophan residue of human serum albumin in the stereospecific binding site for indole and benzodiazepine compounds was investigated by chemical modification. This residue can be selectively modified with 2-hydroxy-5-nitrobenzyl bromide. The modification alters the conformation of the albumin only slightly, as revealed by circular dichroism, fluorescence, and ultraviolet absorption measurements. A decrease in the association constants of L-tryptophan and diazepam of about 30 - 50% and a decrease in the extrinsic Cotton effects of four benzodiazepine derivatives of about 10 - 15% were found as specific effects of the tryptophan modification. The tryptophan modification itself did not change the number of binding sites of diazepam and L-tryptophan. It is suggested that the lone tryptophan residue of human serum albumin is not directly involved in the specific binding site for indole and benzodiazepine compounds. However, the modification alters the properties of the binding site either by an incomplete refolding of the albumin after urea treatment, or a more selective allosteric effect of the modified tryptophan residue.     

复合氨基酸注射液中L-色氨酸含量测定方法的研究

利用色氨酸结构中的吲哚基团在酸性条件下与对二甲氨基苯甲醛的特殊反应 ,在 6 10nm处测定复合氨基酸注射液中L 色氨酸的含量 ,获得满意的结果     

Production L-tryptophan by Escherichia coli cells

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

The reaction of indole with the aminoacrylate intermediate of Salmonella typhimurium tryptophan synthase: observation of a primary kinetic isotope effect with 3-[(2)H]indole

The bacterial tryptophan synthase alpha(2)beta(2) complex catalyzes the final reactions in the biosynthesis of L-tryptophan. Indole is produced at the active site of the alpha-subunit and is transferred through a 25-30 A tunnel to the beta-active site, where it reacts with an aminoacrylate intermediate. Lane and Kirschner proposed a two-step nucleophilic addition-tautomerization mechanism for the reaction of indole with the aminoacrylate intermediate, based on the absence of an observed kinetic isotope effect (KIE) when 3-[(2)H]indole reacts with the aminoacrylate intermediate. We have now observed a KIE of 1.4-2.0 in the reaction of 3-[(2)H]indole with the aminoacrylate intermediate in the presence of monovalent cations, but not when an alpha-subunit ligand, disodium alpha-glycerophosphate (Na(2)GP), is present. Rapid-scanning stopped flow kinetic studies were performed of the reaction of indole and 3-[(2)H]indole with tryptophan synthase preincubated with L-serine, following the decay of the aminoacrylate intermediate at 350 nm, the formation of the quinonoid intermediate at 476 nm, and the formation of the L-Trp external aldimine at 423 nm. The addition of Na(2)GP dramatically slows the rate of reaction of indole with the alpha-aminoacrylate intermediate. A primary KIE is not observed in the reaction of 3-[(2)H]indole with the aminoacrylate complex of tryptophan synthase in the presence of Na(2)GP, suggesting binding of indole with tryptophan synthase is rate limiting under these conditions. The reaction of 2-methylindole does not show a KIE, either in the presence of Na(+) or Na(2)GP. These results support the previously proposed mechanism for the beta-reaction of tryptophan synthase, but suggest that the rate limiting step in quinonoid intermediate formation from indole and the aminoacrylate intermediate is deprotonation.     

A facile means for the identification of indolic compounds from plant tissues

The bulk of indole‐3‐acetic acid (IAA) in plants is found in the form of conjugated molecules, yet past research on identifying these compounds has largely relied on methods that were both laborious and inefficient. Using recent advances in analytical instrumentation, we have developed a simple yet powerful liquid chromatography–mass spectrometry (LC–MS)‐based method for the facile characterization of the small IAA conjugate profile of plants. The method uses the well‐known quinolinium ion (m/z 130.0651) generated in MS processes as a signature with high mass accuracy that can be used to screen plant extracts for indolic compounds, including IAA conjugates. We reinvestigated Glycine max (soybean) for its indoles and found indole‐3‐acetyl‐trytophan (IA‐Trp) in addition to the already known indole‐3‐acetyl‐aspartic acid (IA‐Asp) and indole‐3‐acetyl‐glutamic acid (IA‐Glu) conjugates. Surprisingly, several organic acid conjugates of tryptophan were also discovered, many of which have not been reported in planta before. These compounds may have important physiological roles in tryptophan metabolism, which in turn can affect human nutrition. We also demonstrated the general applicability of this method by identifying indolic compounds in different plant tissues of diverse phylogenetic origins. It involves minimal sample preparation but can work in conjunction with sample enrichment techniques. This method enables quick screening of IAA conjugates in both previously characterized as well as uncharacterized species, and facilitates the identification of indolic compounds in general.     

Several known indole compounds are not important precursors of direct mutagenic N-nitroso compounds in green cabbage

In this study we investigated the role of indole-3-acetonitrile, indole-3-carbinol, indole and tryptophan in the formation of N-nitroso compounds in green cabbage extracts. Green cabbage extracts were separated by gel permeation chromatography. Fractions were treated with nitrite, tested for mutagenicity and analysed for total N-nitroso content. Fractions in which spiked indole-3-acetonitrile, indole-3-carbinol, indole and tryptophan eluted appeared to be low in mutagenic activity and contained relatively small amounts of N-nitroso compounds. To detect indole compounds other than the ones used in the gel permeation chromatography experiments, high-performance liquid chromatography and gas chromatography-mass spectrometry analyses were performed of green cabbage extracts. Indole-3-carboxaldehyde was found to be the most commonly occurring indole compound, but it did not show direct mutagenic activity upon nitrite treatment. Indole-3-acetonitrile was the second most common compound; although it was mutagenic after nitrite treatment, its contribution to the mutagenicity of nitrite-treated green cabbage was roughly estimated to be only 2%. No other indole compounds were detected. From this study we conclude that neither the tested indole compounds nor indole-3-carboxaldehyde play a significant role in the formation of direct mutagenic N-nitroso compounds in nitrite-treated green cabbage extracts.     

The tryptophan aminotransferase Tam1 catalyses the single biosynthetic step for tryptophan-dependent pigment synthesis in Ustilago maydis

Tryptophan is a precursor for many biologically active secondary metabolites. We have investigated the origin of indole pigments first described in the pityriasis versicolor-associated fungus Malassezia furfur . Some of the identified indole pigments have properties potentially explaining characteristics of the disease. As M. furfur is not amenable to genetic manipulation, we used Ustilago maydis to investigate the pathway leading to pigment production from tryptophan. We show by high-performance liquid chromatography, mass spectrometry and nuclear magnetic resonance analysis that the compounds produced by U. maydis include those putatively involved in the etiology of pityriasis versicolor. Using a reverse genetics approach, we demonstrate that the tryptophan aminotransferase Tam1 catalyses pigment biosynthesis by conversion of tryptophan into indolepyruvate. A forward genetics approach led to the identification of mutants incapable of producing the pigments. These mutants were affected in the sir1 gene, presumably encoding a sulphite reductase. In vitro experiments with purified Tam1 showed that 2-oxo 4-methylthio butanoate serves as a substrate linking tryptophan deamination to sulphur metabolism. We provide the first direct evidence that these indole pigments form spontaneously from indolepyruvate and tryptophan without any enzymatic activity. This suggests that compounds with a proposed function in M. furfur -associated disease consist of indolepyruvate-derived spontaneously generated metabolic by-products.     

Evolution of the biosynthesis of the branched-chain amino acids

The origin of the biosynthetic pathways for the branched-chain amino acids cannot be understood in terms of the backwards development of the present acetolactate pathway because it contains unstable intermediates. We propose that the first biosynthesis of the branched-chain amino acids was by the reductive carboxylation of short branched chain fatty acids giving keto acids which were then transaminated. Similar reaction sequences mediated by nonspecific enzymes would produce serine and threonine from the abundant prebiotic compounds glycolic and lactic acids. The aromatic amino acids may also have first been synthesized in this way, e.g. tryptophan from indole acetic acid. The next step would have been the biosynthesis of leucine from -ketoisovaleric acid. The acetolactate pathway developed subsequently. The first version of the Krebs cycle, which was used for amino acid biosynthesis, would have been assembled by making use of the reductive carboxylation and leucine biosynthesis enzymes, and completed with the development of a single new enzyme, succinate dehydrogenase. This evolutionary scheme suggests that there may be limitations to inferring the origins of metabolism by a simple back extrapolation of current pathways.     

Modification of voltage-dependent gating of potassium channels by free form of tryptophan side chain.

Indole constitutes a major component of the side chain of the amino acid tryptophan. Application of indole slows activation of voltage-dependent potassium channels and reduces steady-state conductance in a voltage- and concentration-dependent manner. The steep concentration dependence indicates that multiple indole molecules may interact with the channel. Indole does not noticeably change the unitary conductance or the mean open duration, however, it accelerates off-gating currents without altering on-gating currents. These properties of the modification of channel gating induced by indole are consistent with a model in which indole binds independently to every subunit of the channel complex to prevent the final concerted transition to the open state. We suggest that exogenously applied indole and side-chains of the tryptophan residues of the channel protein involved in activation may compete for the same effector position and that indole might be useful as a probe to study functional roles of tryptophan residues.     

Physiological studies of biosynthetic indole excretion in Bacillus alvei

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

Formation of diastereoisomeric 3a-hydroxypyrroloindoles from a tryptophan residue analog mediated by iron (II)-EDTA and L-ascorbate

Oxygenation of a tryptophan residue analog by ascorbate in the presence of catalytic amounts of iron(II) and ethylenediaminetetraacetic acid (EDTA) has been studied. Under physiological conditions, reaction of the tryptophan derivative (N-t-butoxycarbonyl-L-tryptophan) with Fe(II)-EDTA and ascorbate resulted mainly in the oxygenation of the indole moiety of the substrate. In this reaction, cis and trans diastereoisomeric alcohols 3a-hydroxy-1-t-butoxycarbonyl-1,2,3,3a,8,8a-hexahydropyrrolo[2,3- b]indoles have been successfully identified in the metal-catalyzed free radical oxidation of indole compounds. Hydroxylation at C-5 and C-6 and a ring opening reaction between C-2 and C-3 have also been confirmed. The reaction of Fe(II)-EDTA/ascorbate with the tryptophan derivative was apparently nonselective with regard to position and was significantly suppressed by the hydroxyl radical scavengers (mannitol and dimethylsulfoxide), suggesting the participation of the hydroxyl radical as the actual oxidizing species.     

Tryptophan Metabolism and Kynureninase Induction in Mutants of Neurospora crassa Resistant to 4-Methyl-Tryptophan

Mutants of Neurospora crassa that are resistant to 4-methyl-tryptophan were found to differ in ability to synthesize kynureninase in the presence of the inducers kynurenine, 3-OH-kynurenine, N-formyl-kynurenine, tryptophan, and indole. One strain (mtr26), although incapable of accumulating intracellular pools of these compounds, showed induced synthesis of kynureninase, whereas the second (mtr21) could neither accumulate nor be induced by them. Strain mtr21, with the suppressor su(mtr), could not be induced by indole but was induced by tryptophan and kynurenine derivatives. These results suggest that the mtr mutation, in addition to altering the ability of these strains to concentrate tryptophan and its metabolites, may have some effect on either the intracellular distribution of tryptophan or directly on the synthesis of kynureninase.