Potential role of tryptophan and chloride in the inhibition of human myeloperoxidase

Myeloperoxidase (MPO) binds H2O2 in the absence and presence of chloride (Cl-) and catalyzes the formation of potent oxidants through 1e(-) and 2e(-) oxidation pathways. These potent oxidants have been implicated in the pathogenesis of various diseases including atherosclerosis, asthma, arthritis, and cancer. Thus, inhibition of MPO and its by-products may have a wide application in biological systems. Using direct rapid kinetic measurements and H2O2-selective electrodes, we show that tryptophan (Trp), an essential amino acid, is linked kinetically to the inhibition of MPO catalysis under physiological conditions. Trp inactivated MPO in the absence and presence of plasma levels of Cl(-), to various degrees, through binding to MPO, forming the inactive complexes Trp-MPO and Trp-MPO-Cl, and accelerating formation of MPO Compound II, an inactive form of MPO. Inactivation of MPO was mirrored by the direct conversion of MPO-Fe(III) to MPO Compound II without any sign of Compound I accumulation. This behavior indicates that Trp binding modulates the formation of MPO intermediates and their decay rates. Importantly, Trp is a poor substrate for MPO Compound II and has no role in destabilizing complex formation. Thus, the overall MPO catalytic activity will be limited by: (1) the dissociation of Trp from Trp-MPO and Trp-MPO-Cl complexes, (2) the affinity of MPO Compound I toward Cl(-) versus Trp, and (3) the slow conversion of MPO Compound II to MPO-Fe(III). Importantly, Trp-dependent inhibition of MPO occurred at a wide range of concentrations that span various physiological and supplemental ranges.     

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.     

Origin of the different pH activity profile in two homologous ketosteroid isomerases

Two homologous Delta5-3-ketosteroid isomerases from Comamonas testosteroni (TI-WT) and Pseudomonas putida biotype B (PI-WT) exhibit different pH activity profiles. TI-WT loses activity below pH 5.0 due to the protonation of the conserved catalytic base, Asp-38, while PI-WT does not. Based on the structural analysis of PI-WT, the critical catalytic base, Asp-38, was found to form a hydrogen bond with the indole ring NH of Trp-116, which is homologously replaced with Phe-116 in TI-WT. To investigate the role of Trp-116, we prepared the F116W mutant of TI-WT (TI-F116W) and the W116F mutant of PI-WT (PI-W116F) and compared kinetic parameters of those mutants at different pH levels. PI-W116F exhibited significantly decreased catalytic activity at acidic pH like TI-WT, whereas TI-F116W maintained catalytic activity at acidic pH like PI-WT and increased the kcat/Km value by 2.5- to 4.7-fold compared with TI-WT at pH 3.8. The crystal structure of TI-F116W clearly showed that the indole ring NH of Trp-116 could form a hydrogen bond with the carboxyl oxygen of Asp-38 like that of PI-WT. The present results demonstrate that the activities of both PI-WT and TI-F116W at low pH were maintained by a tryptophan, which was able not only to lower the pKa value of the catalytic base but also to increase the substrate affinity. This is one example of the strategy nature can adopt to evolve the diversity of the catalytic function in the enzymes. Our results provide insight into deciphering the molecular evolution of the enzyme and creating novel enzymes by protein engineering.     

The chemical modification of the essential groups of beta-N-acetyl-D-glucosaminidase from Turbo cornutus Solander

The chemical modification of beta-N-acetyl-D-glucosaminidase (EC3.2.1.30) from Turbo cornutus Solander has been first studied. The results demonstrate that the sulfhydryl group of cysteine residues and the hydroxyl group of serine residues are not essential to the enzyme's function. The modification of indole group of tryptophan of the enzyme by N-bromosuccinimide (NBS) can lead to the complete inactivation, accompanying the absorption decreasing at 278 nm and the fluorescence intensity quenching at 335 nm, indicating that tryptophan is essential residue to the enzyme. The modification of amino group of lysine residue by formaldehyde and trinitrobenzenesulfonic acid also inactivates the enzyme completely. The results show that lysine and tryptophan are probably situated in the active site of the enzyme. The modification of the imidazole residue and carboxyl group leads to inactivate incompletely, indicating they are not the composing groups of the enzyme active center, and they are essential for maintaining the enzyme's conformation which is necessary for the catalytic activity of the enzyme.     

STRUCTURAL REQUIREMENTS FOR AMINO ACID INHIBITION OF Na+-DEPENDENT PROLINE UPTAKE BY RAT BRAIN SYNAPTOSOMES

Abstract— The structural requirements for amino acid inhibition of Na⁺-dependent proline uptake by rat brain synaptosomal fractions were investigated. It is shown that the amino group has to be in the α-position to strongly inhibit proline uptake. Hydroxyamino acids are less potent inhibitors than the parent amino acids. Amino acids with net positive or negative charges on their molecules exert no effect, whereas elimination of the net charge results in compounds with profound inhibitory effects. Blocking of the carboxyl group reduces the inhibition, but does not abolish it. Since acetylation of the α-amino group results in elimination of the inhibitory effect whereas N -methylation does not, it is concluded that in the interaction of an amino acid with the proline transport site the positive charge on the amino group plays the most critical role.     

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.     

Carbon monoxide-releasing molecule 3 inhibits myeloperoxidase (MPO) and protects against MPO-induced vascular endothelial cell activation/dysfunction

Polymorphonuclear leukocyte (PMN)-derived myeloperoxidase (MPO) contributes to the pathophysiology of numerous systemic inflammatory disorders through: (1) direct peroxidation of targets and (2) production of strong oxidizing compounds, e.g., hypohalous acids, particularly hypochlorous acid, which furthers oxidant damage and contributes to the propagation of inflammation and tissue injury/dysfunction. Carbon monoxide-releasing molecules (CORMs) offer potent anti-inflammatory effects; however, the mechanism(s) of action is not fully understood. This study assessed the potential of MPO activity inhibition by a water-soluble CORM, CORM-3. To this end, we used in vitro assays to study CORM-3-dependent modulation of MPO activity with respect to: (1) the inhibition of MPO’s catalytic activity generally and (2) the specific inhibition of MPO’s peroxidation and halogenation (i.e., production of hypochlorous acid) reactions. Further, we employed primary human umbilical vein endothelial cells (HUVECs) to investigate MPO-dependent cellular activation and dysfunction by measuring intracellular oxidant stress (DHR-123 oxidation) and HUVEC permeability (flux of Texas red–dextran), respectively. The results indicate that CORM-3 significantly inhibits MPO activity as well as MPO’s peroxidation and hypohalous acid cycles specifically (p<0.05 vs uninhibited MPO). In addition, CORM-3 significantly decreases PMN homogenate- or rhMPO-induced intracellular DHR-123 oxidation in HUVECs and rhMPO-induced HUVEC monolayer permeability (p<0.05 vs untreated). In all assays the inactivated CORM-3 was significantly less effective than CORM-3 (p<0.05). Taken together our findings indicate that CORM-3 is a novel MPO inhibitor and mitigates inflammatory damage at least in part through a mechanism involving the inhibition of neutrophilic MPO activity.     

Naturally-occurring tetrahydro-β-carboline alkaloids derived from tryptophan are oxidized to bioactive β-carboline alkaloids by heme peroxidases

β-Carbolines are indole alkaloids that occur in plants, foods, and endogenously in mammals and humans, and which exhibit potent biological, psychopharmacological and toxicological activities. They form from naturally-occurring tetrahydro-β-carboline alkaloids arising from tryptophan by still unknown way and mechanism. Results in this research show that heme peroxidases catalyzed the oxidation of tetrahydro-β-carbolines (i.e. 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid and 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid) into aromatic β-carbolines (i.e. norharman and harman, respectively). This oxidation followed a typical catalytic cycle of peroxidases through redox intermediates I, II, and ferric enzyme. Both, plant peroxidases (horseradish peroxidase, HRP) and mammalian peroxidases (myeloperoxidase, MPO and lactoperoxidase, LPO) catalyzed the oxidation in an efficient manner as determined by kinetic parameters (V_MAX and K_M). Oxidation of tetrahydro-β-carbolines was inhibited by peroxidase inhibitors such as sodium azide, ascorbic acid, hydroxylamine and excess of H₂O₂. The formation of aromatic β-carbolines by heme peroxidases can help to explain the presence and activity of these compounds in biological systems.     

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.     

Indolicidin,a 13-residue basic antimicrobial peptide rich in tryptophan and proline,interacts with Ca(2+)-calmodulin

Indolicidin, ILPWKWPWWPWRR-NH(2), a short 13-residue antimicrobial and cytolytic peptide characterized from bovine neutrophils, has the calmodulin-recognition 1-5-10 hydrophobic pattern (indicated by amino acids in bold), is cationic, and thereby fulfills the requirements to interact with calmodulin. Hence, we have investigated the calmodulin-binding properties of indolicidin. Indolicidin interacted with calmodulin with fairly high affinity in a Ca(2+)-dependent manner. However, when bound, the peptide did not adopt helical conformation. Indolicidin also inhibited calmodulin-stimulated phosphodiesterase activity with IC(50) values in the nanomolar range. Replacement of either the proline residues of indolicidin with alanines or tryptophan residues with phenylalanines did not affect binding to calmodulin. However, these replacements had distinctive effects on the conformations of the bound peptides. While the alanine analog of indolicidin adopted predominantly alpha-helical conformation, the phenylalanine analog remained largely unordered. Differences in the ability of these analogs to inhibit the calmodulin-stimulated phosphodiesterase activity were observed. While the alanine analog was capable of inhibiting the activity with IC(50) values comparable to that of indolicidin, the phenylalanine analog did not inhibit the activity. Our results indicate that ability to adopt amphiphilic alpha-helical structure is not a prerequisite for binding to calmodulin and also binding does not necessarily result in inhibition of calmodulin-stimulated enzyme activities.     

The structural basis for the interaction between L-tryptophan and the Escherichia coli trp aporepressor

We have employed equilibrium dialysis to help study the mechanism by which the unliganded Escherichia coli trp aporepressor is activated by L-tryptophan to the liganded trp repressor. By measuring the relative affinity of L-tryptophan and various tryptophan analogues for the co-repressor's binding site, we have estimated the extent to which each of the functional groups of L-tryptophan contributes to the liganding process and discuss their role in the context of the crystal structures of the trp repressor and aporepressor. We have found that the indole ring and alpha carboxyl group of L-tryptophan are mainly responsible for its affinity to the aporepressor. The alpha amino group, however, has a small negative contribution to the affinity of L-tryptophan for the aporepressor which may be associated with its essential role in operator-specific binding.     

Interaction of indole derivatives and tryptophan peptides with interfaces of sodium dodecyl sulfate micelles.

The free energies of transfer for indole and tryptophan derivatives and pentapeptides having single tryptophan residues from aqueous to sodium dodecyl sulfate (SDS) micellar phases have been systematically studied using the conventional method of ultraviolet absorption spectrophotometry. The free energies for the position isomers of methyl indoles varied depending on the substitution positions. Thus, the contribution of the methyl group to the binding affinity of the 4-methyl indole to the micelle was about twice that of the 2- and 7-methyl indoles. The free energy changes with the introduction of halogen groups to the indole rings were correlated to the nonpolar water-accessible surface area (DeltaA(np)) of the halogen moieties, which were regarded as hydrophobic. The relationships followed straight lines passing through the origins. Position dependence having tendencies similar to the methyl indoles was observed among the magnitudes of the slopes of the straight lines. These results strongly suggest that the indole rings of the derivatives residing in the micellar interface regions direct their imino moieties --NH-- toward the micellar surfaces. Experiments using model tryptophan pentapeptides showed that the magnitude of free energy change per methylene unit of an alkyl amino acid residue in the pentapeptide increased with elongation of the alkyl moiety and was not a constant value as reported for various alkyl compounds. When the peptides distribute to the SDS micelles, the peptide backbones are anchored in aqueous phases and the amino acid side chains in the interfaces extend their alkyl groups toward the micellar centers. Thus, the free energy changes can be connected to the positions of the alkyl groups of the amino acid residues in the micelles.     

Melatonin analogue new indole hydrazide/hydrazone derivatives with antioxidant behavior: Synthesis and structure–activity relationships

Melatonin (MLT) is a hormone produced in the brain by the pineal gland, from the amino acid tryptophan. It is also an antioxidant hormone with a particular role in the protection of nuclear and mitochondrial DNA. In recent years, many physiological properties of MLT have been described resulting in much attention in the development of synthetic compounds possessing the indole ring. Sixteen MLT analogue indole hydrazide/hydrazone derivatives were synthesized and in vitro antioxidant activity was investigated. Most of the compounds showed significantly higher activity than MLT at 10^{? 3} M and 10^{? 4} M concentrations.     

Chemical modification of xylanase from alkalothermophilic Bacillus species: evidence for essential carboxyl group

The role of carboxyl group in the catalytic action of xylanase (M_r 35 000) from an alkalothermophilic Bacillus sp. was delineated through iinetic and chemical modification studies using Woodward's Reagent K. The kinetics of inactivation indicated that one carboxyl residue was essential for the xylanase activity with a second order rate constant of 3300 M⁻¹ min⁻¹. The spectrophotometric analysis at 340 nm revealed that the inhibition was correlated with modification of 24 carboxyl residues. In the presence of protecting ligand, modification of one carboxyl group was prevented. The pH profile showed apparent pK values of 5.2 and 6.4 for the free enzyme and 4.9 and 6.9 for enzyme-substrate complex. The pH dependence of inactivation was consistent with the modification of carboxyl group. The kinetic analysis of the modified enzyme showed similar K_m and lower k_cat values than the native enzyme indicating that catalytic hydrolysis and not the substrate binding was affected by chemical modification. The chemical modification of xylanase from alkalothermophilic Bacillus revealed the presence of tryptophans in the active site (Dehspande, V, Hinge, J. and Rao, M. (1990) Biochim. Biophys. Acta 1041, 172–177). This finding and present studies demonstrated the experimental evidence for the participation of carboxyl as well as tryptophan groups as essential residues of xylanase from alkalothermophilic Bacillus sp.     

雷公藤植物内生Micromonospora sp. M66生产的一组吲哚生物碱

【目的】研究稀有放线菌——雷公藤内生小单孢菌(Micromonospora sp.M66)的次级代谢产物,为微生物药物或农用生物制剂开发提供结构多样的化合物资源。【方法】利用薄层层析、正(反)相硅胶柱层析、凝胶层析、液相色谱等技术对M66菌株中次级代谢产物进行分离纯化,利用波谱技术对化合物进行结构鉴定。【结果】最终分离纯化了7个单体化合物,结合质谱与核磁技术对这7个化合物进行了结构解析和鉴定,它们属于一组吲哚生物碱。化合物2是重要的植物生长调节剂,化合物3对淋巴细胞性白血病细胞P388、枯草芽孢杆菌和酿酒酵母的增殖有抑制作用,化合物6对金黄色葡萄球菌有很好的抑制作用。【结论】化合物3-7首次从小单胞菌中鉴定出来,表明该小单孢菌具有较强的利用吲哚或色氨酸合成次级代谢产物的能力和挖掘生物碱类药物的潜力。     

Expression and Deletion Mutagenesis of Tryptophan Hydroxylase Fusion Proteins: Delineation of the Enzyme Catalytic Core

Abstract: cDNAs encoding the full-length sequence for tryptophan hydroxylase, and deletion mutants consisting of the regulatory (amino acids 1–98) or catalytic (amino acids 99–444) domains of the enzyme, were cloned and expressed as glutathione S -transferase fusion proteins in E. coli . The recombinant fusion proteins could be purified to near homogeneity within minutes by affinity chromatography on glutathione-agarose. The full-length enzyme and the catalytic core expressed very high levels of tryptophan hydroxylase activity. The regulatory domain was devoid of activity. The full-length enzyme and the catalytic core, while adsorbed to glutathione-agarose beads, obeyed Michaelis-Menten kinetics, and the kinetic properties of each recombinant enzyme for cofactor and substrate compared very closely to native, brain tryptophan hydroxylase. Both active forms of the glutathione S -transferase-tryptophan hydroxylase fusion proteins had strict requirements for ferrous iron in catalysis and expressed much higher levels of activity ( V _max) than the brain enzyme. Analysis of full-length tryptophan hydroxylase and the catalytic core by molecular sieve chromatography under nondenaturing conditions revealed that each fusion protein behaved as a tetrameric species. These results indicate that a truncated tryptophan hydroxylase, consisting of amino acids 99–444 of the full-length enzyme, contains the sequence motifs needed for subunit assembly. Both wild-type tryptophan hydroxylase and the catalytic core are expressed as apoenzymes which are converted to holoenzymes by exogenous iron. The tryptophan hydroxylase catalytic core is also as active as the full-length enzyme, suggesting the possibility that the regulatory domain exerts a suppressive effect on the catalytic core of tryptophan hydroxylase.     

Characteristics of the Amino Acid Transport System in the Mucosal Border of Rabbit Ileum

The specificity of the neutral amino acid transport system in the brush border was examined by studying the ability of amino acid analogues to inhibit the unidirectional influx of phenylalanine from mucosal solution into the cells. Effects were evaluated in terms of the affinity of various substrates for the amino acid site in the transport system. The affinity of amino acids for the site was proportional to the number of carbon atoms in the side chain. Electron-withdrawing substituents in the ring of phenylalanine increased affinity and electron-releasing groups decreased affinity. Removal of the α-amino group from phenylalanine decreased affinity by a factor of approximately 50 and removal of the carboxyl group decreased affinity 12-fold. Effects on affinity of variations in the side chain of the amino acid can be comparable in magnitude to that of the carboxyl group. The effect of sodium ion on the transport system appears to be similar for all compounds tested.     

Melatonin analogue new indole hydrazide/hydrazone derivatives with antioxidant behavior: synthesis and structure-activity relationships

Melatonin (MLT) is a hormone produced in the brain by the pineal gland, from the amino acid tryptophan. It is also an antioxidant hormone with a particular role in the protection of nuclear and mitochondrial DNA. In recent years, many physiological properties of MLT have been described resulting in much attention in the development of synthetic compounds possessing the indole ring. Sixteen MLT analogue indole hydrazide/hydrazone derivatives were synthesized and in vitro antioxidant activity was investigated. Most of the compounds showed significantly higher activity than MLT at 10(-3) M and 10(-4) M concentrations.     

Synthesis and DNA binding properties of tryptophan linked monocationic netropsin analogues

One tryptophan (Trp-Net) and two tryptophan (Trp-Trp-Net) residues have been linked to the amino terminus of a minor groove binding Netropsin analogue. DNA metling measurements indicate that Trp-Trp-Net binds significantly stronger than Trp-Net to double helical DNA. Both compounds induce helix extension as measured by changes in DNA viscosity indicating the possibility of intercalation of the tryptophan indole ring.     

Characterization of thyroxine-albumin binding using high-performance affinity chromatography II. Comparison of the binding of thyroxine,triiodothyronines and related compounds at the warfarin and indole sites of human serum albumin

High-performance affinity chromatography was used to examine the binding of thyroid hormones and related compounds at the warfarin and indole sites of human serum albumin (HSA). This was studied by continuously applying l-triiodothyronine (l-T₃), l-reverse triiodothyronine (l-rT₃) or structural analogs of these compounds to an immobilized HSA column while making injections of site-specific probe molecules (i.e. R-warfarin and l-tryptophan). The results were compared with those obtained previously for l-thyroxine (l-T₄). Equilibrium association constants and thermodynamic parameters measured by this approach showed good agreement with previous models reported for l-T₄ and l-T₃ at their high-affinity sites on HSA. This data confirmed that the phenol groups of l-T₄ and l-T₃ played a significant role in the binding of these compounds at the indole site. Work performed at the warfarin site and with other solutes (e.g. l-rT₃) indicated that additional factors, such as interactions through the thyronine backbone or terminal amine and carboxyl groups of these compounds, could also be involved in the binding of thyroid hormones to HSA.