The binding of amino acids to the herbicide 2,4-dichlorophenoxy acetic acid

Summary. The interaction of amino acids with the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was studied by charge-transfer chromatography carried out on diatomaceous layers covered with different amount of 2,4-D and the effect of salts on the strength of interaction was elucidated. It was established that Arg, His, Lys, Orn, Phe and Trp binds to 2,4-D, the binding process is of saturation character. Principal component analysis proved that the concentration of 2,4-D exerts the highest impact on the interaction and the effect of salts is of secondary importance. The results suggest that these amino acid residues may account for the binding of 2,4-D to proteins and can play a considerable role in the detoxification processes by forming conjugates with 2,4-D. Received April 10, 1998, Accepted September 15, 1998     

Fluorescence quenching method for the determination of carbazochrome sodium sulfonate with aromatic amino acids

In Britton‐Robinson (BR) buffer medium (pH 3.3), carbazochrome sodium sulfonate (CSS) can react with some aromatic amino acids such as tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe) to form a 1:1 complex by electrostatic attraction, aromatic stacking interaction and Van der Waals' force, resulting in fluorescence quenching of these amino acids. Maximum quenching wavelengths were located at 352 nm (CSS‐Trp system), 303 nm (CSS‐Tyr system) and 284 nm (CSS‐Phe system), respectively. The fluorescence quenching value (ΔF) was proportional to the concentration of CSS in a certain range. The fluorescence quenching method for the determination of CSS showed high sensitivity, with detection limits of 31.3 ng/mL (CSS‐Trp system), 44.6 ng/mL (CSS‐Tyr system) and 315.0 ng/mL (CSS‐Phe system), respectively. The optimum conditions of the reaction conditions and the effect of coexisting substances were investigated and results showed that the method had good selectivity. The method was successfully applied for the rapid determination of CSS in blood and urine samples. Based on the bimolecular quenching constant K_q, the effect of temperature and Stern‐Volmer plots, this study showed that quenching of fluorescence of amino acids by CSS was a static quenching process. Copyright © 2012 John Wiley & Sons, Ltd.     

The depletion of tryptophan and serotonin in the brain of developing hyperphenylalaninemic rats is abolished by the additional administration of lysine

In suckling hyperphenylalaninemic (hyper-Phe) rats, all essential amino acids including tryptophan are depleted in the blood. The inadequate supply of Trp to the developing brain leads to a decline of Trp, of serotonin, and of 5-hydroxyindoleacetic acid. The exhaustion of amino acids in both blood and brain can be restored by administration of Lys. Even though Phe is still elevated in blood and brain, Trp, serotonin and 5-hydroxyindoleacetic acid, are no longer depleted in the brain. This observation contradicts the idea that the serotonin deficit in the developing hyper-Phe brain is caused by competitive uptake inhibition of tryptophan or by the interference of Phe metabolites with the formation of serotonin. Increased accumulation of all large neutral amino acids in peripheral tissues and an impeded intestinal amino acid transport in suckling hyper-Phe rats appear to be responsible for the deficient supply of other amino acids, including Trp, to the developing brain. The availability of Lys for developing extraintestinal tissues seems to be involved in the regulation of intestinal amino acid transport.     

Analysis of the interactions of sulfur‐containing amino acids in membrane proteins

The interactions of Met and Cys with other amino acid side chains have received little attention, in contrast to aromatic–aromatic, aromatic–aliphatic or/and aliphatic–aliphatic interactions. Precisely, these are the only amino acids that contain a sulfur atom, which is highly polarizable and, thus, likely to participate in strong Van der Waals interactions. Analysis of the interactions present in membrane protein crystal structures, together with the characterization of their strength in small‐molecule model systems at the ab‐initio level, predicts that Met–Met interactions are stronger than Met–Cys ≈ Met–Phe ≈ Cys–Phe interactions, stronger than Phe–Phe ≈ Phe–Leu interactions, stronger than the Met–Leu interaction, and stronger than Leu–Leu ≈ Cys–Leu interactions. These results show that sulfur‐containing amino acids form stronger interactions than aromatic or aliphatic amino acids. Thus, these amino acids may provide additional driving forces for maintaining the 3D structure of membrane proteins and may provide functional specificity.     

The Arabidopsis phenylalanine insensitive growth mutant exhibits a deregulated amino acid metabolism

Amino acids and amino acid analogs have been used in numerous genetic screens to isolate mutants deficient in amino acid biosynthetic pathways or in the regulation of amino acid metabolism. Several of these mutants exhibit relaxed feedback control of branched amino acid biosynthetic pathways and are thus resistant to accumulation of pathway end products. For example, feedback-regulated enzymes of the shikimate pathway are anthranilate synthase on the branch leading to Trp and chorismate mutase on the branch leading to Phe and Tyr. A feedback-insensitive mutant of anthranilate synthase alpha, trp5-1, is resistant to toxic Trp analogs. Mutants resistant to Phe have not previously been reported, and this article describes the isolation of the recessive Arabidopsis Phe insensitive growth mutant pig1-1 by a forward genetic screen. pig1-1 was not only tolerant to Phe, Tyr, and Trp, but also to other, not biosynthetically related amino acids. Amino acid contents in pig1-1 were significantly elevated with respect to wild-type controls but, in contrast to the wild type, dramatically decreased when plants were supplemented with 2 mm Phe. Protein contents were similar in the mutant and the wild type at all tested conditions. Phe catabolism was similar to the wild type in pig1-1 roots but was significantly increased in pig1-1 shoots. Phenylalanine uptake into the root, its root-to-shoot translocation, and Phe and phenylpropanoid contents were unaltered in pig1-1, indicating that pig1-1 is not affected in amino acid translocation or the shikimate pathway. Instead, the response of pig1-1 toward amino acid feeding indicates that amino acid metabolism is generally deregulated in pig1-1.     

烧伤患者血浆和红细胞游离氨基酸变化及输注氨基酸对其变化的影响

动态测定烧伤患者血浆及红细胞内游离氨基酸的含量 ,探讨输入外源性氨基酸后对血及红细胞内游离氨基酸的影响。以日立 835— 5 0型氨基酸自动分析仪测定烧伤患者血浆及红细胞内游离氨基酸含量。结果发现烧伤患者血浆总游离氨基酸浓度从伤后到 2 1天均显著降低 (P <0 .0 5～ 0 .0 1) ;赖、苯丙和苯丙 酪氨酸比值显著升高 (P <0 .0 5～ 0 .0 1) ;色、组、精、丙、甘、苏、脯和丝氨酸比值显著降低 (P <0 .0 5～ 0 .0 1) ;缬、亮、异亮、酪、胱和支链氨基酸伤后早期降低。烧伤患者红细胞内总游离氨基酸含量不同程度降低 ,其中 1、3、7天降低显著 (P <0 .0 5～ 0 .0 1) ;红细胞内苯丙和苯丙 酪氨酸比值未见显著性升高 ;色、蛋、精、脯氨酸含量很低或基本未测出。输注复合氨基酸注射液后未能显著改善患者血及红细胞内游离氨基酸含量。结果提示烧伤患者红细胞内游离氨基酸含量的变化趋势与血浆游离氨基酸变化趋势基本一致 ;烧伤后红细胞内苯丙氨酸及苯丙 酪氨酸比值有别于血浆变化。本研究条件下补充外源性氨基酸未能显著改变烧伤患者血浆及红细胞内游离氨基酸的含量     

Functional and structural consequences of aromatic residue substitutions within the kringle-2 domain of tissue-type plasminogen activator.

Aromatic amino acid residues within kringle domains play important roles in the structural stability and ligand-binding properties of these protein modules. In previous investigations, it has been demonstrated that the rigidly conserved Trp25 is primarily involved in stabilizing the conformation of the kringle-2 domain of tissue-type plasminogen activator (K2tpA), whereas Trp63, Trp74, and Tyr76 function in omega-amino acid ligand binding, and, to varying extents, in stabilizing the native folding of this kringle module. In the current study, the remaining aromatic residues of K2tPA, viz., Tyr2, Phe3, Tyr9, Tyr35, Tyr52, have been subjected to structure-function analysis via site-directed mutagenesis studies. Ligand binding was not significantly influenced by conservative amino acid mutations at these residues, but a radical mutation at Tyr35 destabilized the interaction of the ligand with the variant kringle. In addition, as reflected in the values of the melting temperatures, changes at Tyr9 and Tyr52 generally destabilized the native structure of K2tPA to a greater extent than changes at Tyr2, Phe3, and Tyr35. Taken together, results to date show that, in concert with predictions from the crystal structure of K2tpA, ligand binding appears to rely most on the integrity of Trp63 and Trp74, and aromaticity at Tyr76. With regard to aromatic amino acids, kringle folding is most dependent on Tyr9, Trp25, Tyr52, Trp63, and Tyr76. As yet, no obvious major roles have been uncovered for Tyr2, Phe3, or Tyr35 in K2tpA.     

Modulation of apolipoprotein A-IV lipid binding by an interaction between the N and C termini

Apolipoprotein A-IV (apoA-IV) is a 376-amino acid exchangeable apolipoprotein made in the small intestine of humans. Although it has many proposed roles in vascular disease, satiety, and chylomicron metabolism, there is no known structural basis for these functions. The ability to associate with lipids may be a key step in apoA-IV functionality. We recently identified a single amino acid, Phe(334), which seems to inhibit the lipid binding capability of apoA-IV. We also found that an intact N terminus was necessary for increased lipid binding of Phe(334) mutants. Here, we identify Trp(12) and Phe(15) as the N-terminal amino acids required for the fast lipid binding seen with the F334A mutant. Furthermore, we found that individual disruption of putative amphipathic alpha-helices 3-11 had little effect on lipid binding, suggesting that the N terminus of apoA-IV may be the operational site for initial lipid binding. We also provide three independent pieces of experimental evidence supporting a direct intramolecular interaction between sequences near amino acids 12/15 and 334. This interaction could represent a unique "switch" mechanism by which apoA-IV changes lipid avidity in vivo.     

癌肿与氨基酸代谢的研究

研究了癌肿与氨基酸代谢的关系。这些癌肿包括喉癌HepⅡ细胞 ,急性非淋巴细胞白血病和急性淋巴细胞白血病 ,结果表明 :( 1 )喉癌细胞株培养过程中亮氨酸、赖氨酸、丝氨酸、天冬酰胺、异亮氨酸、甘氨酸以及苏氨酸等水平明显降低 ,而色氨酸水平明显增加 ,说明喉癌细胞的生长繁殖必须依赖以上 7种氨基酸同时释放了色氨酸 ;( 2 )急性非淋巴细胞白血病 (ANLL)患者血浆中的谷氨酸、甘氨酸、亮氨酸、苯丙氨酸、酪氨酸和色氨酸等水平明显升高 ,而苏氨酸、组氨酸、丙氨酸等水平明显降低 ,这些结果与国际报道相一致 ;( 3)经治疗后 ,ANLL患者血浆中甘氨酸、色氨酸和苯丙氨酸等水平明显降低 ,而丙氨酸、组氨酸等水平明显升高 ,表明肿瘤细胞处在无氧代谢。患者经治疗后色氨酸和苯丙氨酸水平降低和组氨酸水平的升高对患者预后是有益的 ;( 4)急性淋巴细胞白血病患者血浆中苯丙氨酸、赖氨酸、色氨酸和酪氨酸水平提高 ,这些氨基酸能促进肿瘤生长 ,而门冬酰胺、谷氨酰胺以及天冬氨酸水平降低 ,说明这 3种氨基酸为肿瘤生长所必须。此外还发现ALL患者外周淋巴细胞中精氨酸水平增加 ,精氨酸对癌肿细胞有直接杀伤作用。     

Hydrophobic interaction between tryptophan and some synthetic nucleosides

The interaction of 29 synthetic nucleosides with tryptophan was studied by charge-transfer reversed-phase thin-layer chromatography and the relative strength of interaction was calculated. In the majority of cases Trp significantly decreased the lipophilicity of the nucleosides. This effect may be due to the interaction between the more hydrophilic Trp and the more lipophilic nucleosides, resulting in charge-transfer complexes of moderate lipophilicity. Stepwise regression analysis proved that the length of the alkyl substituent of nucleosides significantly influences the strength of interaction. Our findings supports the hypothesis that the nucleosides turn toward Trp with their alkyl substituent and the binding is of hydrophobic character.     

Equilibrium of the Cis-Trans Isomerisation of the Peptide Bond with N-alkyl Amino Acids Measured by 2D NMR

Summary The conformationalcis-trans equilibrium around the peptide bond in model tripeptides has been determined by 2D NMR methods (HOHAHA, ROESY). The study was limited to three different N-substituted amino acids in position 2, namely Pro (proline), Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), and N-MePhe (N-methylphenylalanine). In all cases the amino acid in position 1 was tyrosine and in position 3, phenylalanine. The results of our studies show that thecis-trans ratio depends mostly on the configuration of the amino acids forming the peptide bond undergoing thecis-trans isomerisation. The amino acid following the sequence (in position 3) does not have much influence on thecis-trans isomerisation, indicating that there is no interaction of the side chains between these amino acids. The model peptides with the L-Tyr-L-AA-(L-or D-)Phe (where AA is N-substituted amino acid) chiralities give 80–100% more of thecis form in comparison to the corresponding peptides with the D-Tyr-L-AA-(L-or D-)Phe chiralities. These results indicate that the incorporation of N-substituted amino acids in small peptides with the same chirality as the precedent amino acid involved in the peptide bound undergoing thecis/trans isomerisation moves the equilibrium to a significant amount of thecis form.     

Reversed-phase thin-layer chromatography used to study the simultaneous effect of pH and ion strength on the lipophilicity of chlorhexidine

The effect of ion strength and pH value of the eluent on the determination of the lipophilicity of chlorhexidine was studied by reversed-phase thin-layer chromatography. The method has been improved by using various buffers: aqueous solutions of formic, acetic and propionic acids and their sodium salts in different ratios and in various concentrations. Stepwise regression analysis separated the effect of pH value, ion strength and acid type on the lipophilicity of chlorhexidine and proved that the ion strength exerted a higher impact than the pH value did. The effect of alkyl chain length of the acids was of secondary importance.     

Function of conserved aromatic residues in the Gal/GalNAc-glycosyltransferase motif of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 1

UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc transferases), which initiate mucin-type O-glycan biosynthesis, have broad acceptor substrate specificities, and it is still unclear how they recognize peptides with different sequences. To increase our understanding of the catalytic mechanism of GalNAc-T1, one of the most ubiquitous isozymes, we studied the effect of substituting six conserved aromatic residues in the highly conserved Gal/GalNAc-glycosyltransferase motif with leucine on the catalytic properties of the enzyme. Our results indicate that substitutions of Trp302 and Phe325 have little impact on enzyme function and that substitutions of Phe303 and Tyr309 could be made with only limited impact on the interaction(s) with donor and/or acceptor substrates. By contrast, Trp328 and Trp316 are essential residues for enzyme functions, as substitution with leucine, at either site, led to complete inactivation of the enzymes. The roles of these tryptophan residues were further analyzed by evaluating the impact of substitutions with additional amino acids. All evaluated substitutions at Trp328 resulted in enzymes that were completely inactive, suggesting that the invariant Trp328 is essential for enzymatic activity. Trp316 mutant enzymes with nonaromatic replacements were again completely inactive, whereas two mutant enzymes containing a different aromatic amino acid, at position 316, showed low catalytic activity. Somewhat surprisingly, a kinetic analysis revealed that these two amino acid substitutions had a moderate impact on the enzyme's affinity for the donor substrate. By contrast, the drastically reduced affinity of the Trp316 mutant enzymes for the acceptor substrates suggests that Trp316 is important for this interaction.     

Effect of pH and salts on the binding of free amino acids to the corn protein zein studied by thin-layer chromatography

Summary. The interaction of free amino acids with the corn protein zein was studied by thin-layer chromatography carried out on cellulose layers covered with zein and the effect of pH and salts on the strength of interaction was elucidated. Only the binding of Arg, His, Lys, Orn and Trp to zein was verified, other amino acids were not retained. Retention of Arg, His, Lys and Orn decreased linearly with increasing concentration of salts the mobile phase indicating the hydrophilic character of amino acid–zein interaction. Both alkaline and acidic pH influenced the strength of binding. Principal component analysis indicated the different character of the influence of pH and salts on the interaction. The results suggest that these amino acid residues may account for the binding of other peptides and proteins to zein.     

Structural and dynamic characterization of the aromatic amino acids of the human immunodeficiency virus type I nucleocapsid protein zinc fingers and their involvement in heterologous tRNA(Phe) binding: a steady-state and time-resolved fluorescence study.

The steady-state and time-resolved fluorescence properties of two zinc-saturated 18-residue synthetic peptides with the amino acid sequence of the NH2-terminal (NCp7 13-30 F16W, where the naturally occurring Phe was replaced by a Trp residue) and the COOH-terminal (NCp7 34-51) zinc finger domains of human immunodeficiency virus type I nucleocapsid protein were investigated. Fluorescence intensity decay of both Trp 16 and Trp 37 residues suggested the existence of two fully solvent-exposed ground-state classes governed by a C = 2.2 equilibrium constant. The lifetimes of Trp 16 classes differed from those of Trp 37 essentially because of differences in nonradiative rate constants. Arrhenius plots of the temperature-dependent nonradiative rate constants suggested that the fluorescence quenchers involved in both classes and in both peptides were different and the collisional rate of these quenchers with the indole ring was very low, probably because of the highly constrained peptide chain conformation. The nature of the ground-state classes was discussed in relation to 1H nuclear magnetic resonance data. Using Trp fluorescence to monitor the interaction of both peptides with tRNA(Phe) we found that a stacking between the indole ring of both Trp residues and the bases of tRNA(Phe) occurred. This stacking constituted the main driving force of the interaction and modified the tRNA(Phe) conformation. Moreover, the binding of both fingers to tRNA(Phe) was noncooperative with similar site size (3 nucleotide residues/peptide), but the affinity of the NH2-terminal finger domain (K = 1.3 (+/- 0.2) 10(5) M-1) in low ionic strength buffer was one order of magnitude larger than the COOH-terminal one due to additional electrostatic interactions involving Lys 14 and/or Arg 29 residues.     

Structural Bases for the Regulation of CO Binding in the Archaeal Protoglobin from Methanosarcina acetivorans

Studies of CO ligand binding revealed that two protein states with different ligand affinities exist in the protoglobin from Methanosarcina acetivorans (in MaPgb*, residue Cys(E20)101 was mutated to Ser). The switch between the two states occurs upon the ligation of MaPgb*. In this work, site-directed mutagenesis was used to explore the role of selected amino acids in ligand sensing and stabilization and in affecting the equilibrium between the “more reactive” and “less reactive” conformational states of MaPgb*. A combination of experimental data obtained from electronic and resonance Raman absorption spectra, CO ligand-binding kinetics, and X-ray crystallography was employed. Three amino acids were assigned a critical role: Trp(60)B9, Tyr(61)B10, and Phe(93)E11. Trp(60)B9 and Tyr(61)B10 are involved in ligand stabilization in the distal heme pocket; the strength of their interaction was reflected by the spectra of the CO-ligated MaPgb* and by the CO dissociation rate constants. In contrast, Phe(93)E11 is a key player in sensing the heme-bound ligand and promotes the rotation of the Trp(60)B9 side chain, thus favoring ligand stabilization. Although the structural bases of the fast CO binding rate constant of MaPgb* are still unclear, Trp(60)B9, Tyr(61)B10, and Phe(93)E11 play a role in regulating heme/ligand affinity.     

Effect of water coordination on competition between π and non-π cation binding sites in aromatic amino acids: l-phenylalanine, l-tyrosine, and l-tryptophan Li+, Na+, and K+ complexes

Quantum chemistry methods have been applied to charged complexes of the alkali metals Li(+), Na(+), and K(+) with the aromatic amino acids (AAAs) phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp). The geometries of 72 different complexes (Phe·M, Tyr·M, Trp·M, M is Li(+), Na(+), or K(+)) were completely optimized at the B3LYP/6-311+G(d,p) level of density functional theory. The solvent effect on the geometry and stability of individual complexes was studied by making use of a microsolvation model. The interaction enthalpies, entropies, and Gibbs energies of nine different complexes of the systems Phe·M, Tyr·M, and Trp·M (M is Li(+), Na(+), or K(+)) were also determined at the B3LYP density functional level of theory. The calculated Gibbs binding energies of the M(+)-AAA complexes follow the order Phe < Tyr < Trp for all three metal cations studied. Among the three AAAs studied, the indole ring of Trp is the best π donor for alkali metal cations. Our calculations demonstrated the existence of strong cation-π interactions between the alkali metals and the aromatic side chains of the three AAAs. These AAAs comprise about 8% of all known protein sequences. Thus, besides the potential for hydrogen-bond interaction, aromatic residues of Phe, Tyr, and Trp show great potential for π-donor interactions. The existence of cation-π interaction in proteins has also been demonstrated experimentally. However, more complex experimental studies of metal cation-π interaction in diverse biological systems will no doubt lead to more exact validation of these investigations.     

Self‐assembly of short peptides composed of only aliphatic amino acids and a combination of aromatic and aliphatic amino acids

The morphology of structures formed by the self‐assembly of short N‐terminal t‐butyloxycarbonyl (Boc) and C‐terminal methyl ester (OMe) protected and Boc‐deprotected hydrophobic peptide esters was investigated. We have observed that Boc‐protected peptide esters composed of either only aliphatic hydrophobic amino acids or aliphatic hydrophobic amino acids in combination with aromatic amino acids, formed highly organized structures, when dried from methanol solutions. Transmission and scanning electron microscopic images of the peptides Boc‐Ile‐Ile‐OMe, Boc‐Phe‐Phe‐Phe‐Ile‐Ile‐OMe and Boc‐Trp‐Ile‐Ile‐OMe showed nanotubular structures. Removal of the Boc group resulted in disruption of the ability to form tubular structures though spherical aggregates were formed. Both Boc‐Leu‐Ile‐Ile‐OMe and H‐Leu‐Ile‐Ile‐OMe formed only spherical nanostructures. Dynamic light scattering studies showed that aggregates of varying dimensions were present in solution suggesting that self‐assembly into ordered structures is facilitated by aggregation in solution. Fourier transform infrared spectroscopy and circular dichroism spectroscopy data show that although all four of the protected peptides adopt well‐defined tertiary structures, upon removal of the Boc group, only H‐Phe‐Phe‐Phe‐Ile‐Ile‐OMe had the ability to adopt β‐structure. Our results indicate that hydrophobic interaction is a very important determinant for self‐assembly and presence of charged and aromatic amino acids in a peptide is not necessary for self‐assembly. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.