The secondary structure of nucleotidyl-(5' bound to N)-amino acids containing different heterocyclic bases and amino acids]

Nucleotidyl-(5' leads to N)-amino acids containing different heterocycle bases: adenine, guanine, hypoxanthine, cytosine, uracyl, and aromatic amino acids: phenylalanine, tyrosine and tryptophan, have been investigated by proton magnetic resonance and circular dichroism. For all the compounds studied folded conformation have been shown stabilized by hydrophobic interaction in aqueous solution. The comparison of the results of the studied nucleotidyl-(5' leads to N)-amino acids unable us to build four secondary structure types in these very compounds. Phenylalanine and tyrosine derivatives of purine nucleotides can be regarded as the first type, tryptophan derivatives of purine nucleotides as the second type, phenylalanine and tyrosine derivatives of pyrimidine nucleotides as the third type and tryptophan derivatives of pyrimidine nucleotides as the fourth type. For each group of these compounds conformational models have been built. In all these compounds the anti-conformation has been proved to exist.     

Positional effects of monofluorinated phenylalanines on histone acetyltransferase stability and activity

To explore the impact of global incorporation of fluorinated aromatic amino acids on protein function, we investigated the effects of three monofluorinated phenylalanine analogs para-fluorophenylalanine (pFF), meta-fluorophenylalanine (mFF), and ortho-fluorophenylalanine (oFF) on the stability and enzymatic activity of the histone acetyltransferase (HAT), tGCN5. We selected this set of fluorinated amino acids because they bear the same size and overall polarity but alter in side chain shape and dipole direction. Our experiments showed that among three fluorinated amino acids, the global incorporation of pFF affords the smallest perturbation to the structure and function of tGCN5.     

Structure activity of C-terminal modified analogs of Ac-CCK-7.

Previous work indicates that both the C-terminal phenylalanine amide and the tryptophan moieties of cholecystokinin (CCK) are critical pharmacophores for interaction with either the A or B receptor subtypes. We have examined a series of analogs of Ac-CCK-7 [Ac-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe33-NH2] (2) in which the phenyl ring of the C-terminal Phe-NH2 has been modified. Compounds were assessed in binding assays using homogenated rat pancreatic membranes and bovine striatum as the source of CCK-A and CCK-B receptors respectively and for anorectic activity after intraperitoneal administration to rats. Substitution of a number of cycloalkyl or bicyclic aryl moieties for the phenyl ring of phenylalanine33 including cyclopentyl (20), cyclohexyl (21), cyclooctyl (23), 2-(5,6,7,8-tetrahydro)naphthyl (26), 2-naphthyl (27), and 1-naphthyl (29) led to analogs with 10-70 times the anorectic potency of 2. The anorectic activity of 21 was blocked by the specific CCK-A receptor antagonist MK-329. Other bulky aliphatic groups in place of the phenylalanine33 aromatic ring such as isopropyl, 2-adamantyl and cyclohexylmethyl gave derivatives similar to 2 in potency. While most of the new compounds were comparable to CCK in binding assays, 23, 26, 27 and 29 were exceptionally potent with IC50s 10(-11)-10(-14) M in the pancreas. Compounds 23 and 29 were further evaluated for their ability to stimulate amylase secretion and found to have potencies similar to that of CCK. The dissociation between potency in the binding and amylase secretion assays suggests that they may interact with a high affinity binding site which is not coupled to amylase secretion. We conclude that CCK receptors possess a generous hydrophobic pocket capable of accommodating large alkyl groups in place of the side chain of phenylalanine33 and that the pharmacological profile of CCK analogs can be tailored by appropriate exploitation of this finding.     

Docking and binding mode analysis of aryl diketoacids (ADK) at the active site of HCV RNA-dependent RNA polymerase

The pharmacophore-guided docking study of aryl diketoacid (ADK) analogues revealed two distinctive hydrophobic binding sites (a pocket and a groove) around the UTP-binding site of hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp). Interestingly, the hydrophobic binding sites have appropriate shape and size to specifically substituted aromatic rings, which suggests the specific role of substituents on the aromatic ring in determining the binding affinity of the ADK analogue to the active site of the target enzyme. Binding mode analysis of ADK analogues with potent antiviral activity shows highly substituted aromatic rings map well onto the hydrophobic binding sites. For less active compounds, their lack of aromatic substitution and thereby insufficient size can be primarily ascribed to their inability to bind to the hydrophobic binding site. The characteristic binding mode of ADK analogues proposed in this study provides a useful tool in designing a structure–activity relationship study of novel ADK analogues based on various aromatic substituents.     

Protein kinase inhibitor-(6-22)-amide peptide analogs with standard and nonstandard amino acid substitutions for phenylalanine 10. Inhibition of cAMP-dependent protein kinase

The minimal structure in the heat-stable inhibitor protein of cAMP-dependent protein kinase required for a low nanomolar potency of inhibition is the peptide Thr6-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-+ ++Ile22-NH2 (PKI-(6-22)-amide). While primary structural determinants for interaction with the protein kinase are distributed throughout the 17 residues of this peptide, we have previously shown that phenylalanine 10 in the NH2-terminal portion is a particularly important determinant for high affinity binding (Glass, D. B., Cheng, H.-C., Mende-Mueller, L., Reed, J., and Walsh, D. A. (1989) J. Biol. Chem. 264, 8802-8810). To investigate this requirement further, peptide analogs of PKI-(6-22)-amide in which various natural and nonstandard amino acids are substituted for phenylalanine 10 have been synthesized and tested for inhibitory potency against the catalytic subunit of the protein kinase. Consistent with the importance of the hydrophobicity of phenylalanine, an alanine 10 substitution analog exhibited a 270-fold decrease in inhibitory potency, whereas the leucine 10 analog lost only 33-fold in activity as compared to the parent peptide PKI-(6-22)-amide. Peptides containing the spatial conformation analogs D-phenylalanine, homophenylalanine, or phenylglycine were 60-120-fold less potent than the parent peptide. Peptides containing various para-substituted phenylalanines at position 10 were only 5-11-fold less potent. One exception to this was (4'-azidophenylalanine 10)PKI-(6-22)-amide, which was nearly equipotent with the parent inhibitor. The most potent analogs were those peptides containing highly aromatic residues at position 10. The 2'-thienylalanine 10, tryptophan (formyl) 10, tryptophan 10, and the 1'-naphthylalanine 10 analogs were 3-fold less potent, equipotent, slightly more potent, and 4-fold more potent than the parent peptide inhibitor, respectively. We conclude that phenylalanine 10 in PKI-(6-22)-amide, and presumably in the native protein inhibitor, interacts through specific hydrophobic and/or aromatic binding to a hydrophobic pocket or cleft near the active site of the protein kinase.     

Modification of the Phe3 aromatic moiety in delta receptor-selective dermorphin/deltorphin-related tetrapeptides. Effects on opioid receptor binding.

The previously described cyclic delta opioid receptor-selective tetrapeptide H-Tyr-D-Cys-Phe-D-Pen-OH (JOM-13) was modified at residue 3 by incorporation of both natural and unnatural amino acids with varying steric, electronic, and lipophilic properties. Effects on mu and delta opioid receptor binding affinities were evaluated by testing the compounds for displacement of radiolabeled receptor-selective ligands in a guinea pig brain receptor binding assay. Results obtained with the bulky aromatic 1-Nal3 and 2-Nal3 substitutions suggest that the shape of the receptor subsite with which the side chain of the internal aromatic residue interacts differs for delta and mu receptors. This subsite of either receptor can accommodate the transverse steric bulk of the 1-Nal3 side chain but only the delta receptor can readily accept the more elongated 2-Nal3 side chain. Several analogs with pi-excessive heteroaromatic side chains in residue 3 were examined. In general, these analogs display diminished binding to mu and delta receptors, consistent with previous findings for analogs with residue 3 substitutions of modified electronic character. Several analogs with alkyl side chains in residue 3 were also examined. While delta receptor binding affinity is severely diminished with Val3, Ile3, and Leu3 substitutions, Cha3 substitution is very well tolerated, indicating that, contrary to the widely held belief, an aromatic side chain in this portion of the ligand is not required for delta receptor binding. Where possible, comparison of results in this delta-selective tetrapeptide series with those reported for analogous modification in the cyclic delta-selective pentapeptide [D-Pen2, D-Pen5]enkephalin (DPDPE) and linear pentapeptide enkephalins reveals similar trends.     

Analysis of tryptophan‐rich region in Clostridium septicum alpha‐toxin involved with binding to glycosylphosphatidylinositol‐anchored proteins

Clostridium septicum alpha‐toxin has a unique tryptophan‐rich region (³⁰²NGYSEWDWKWV³¹²) that consists of 11 amino acid residues near the C‐terminus. Using mutant toxins, the contribution of individual amino acids in the tryptophan‐rich region to cytotoxicity and binding to glycosylphosphatidylinositol (GPI)‐anchored proteins was examined. For retention of maximum cytotoxic activity, W307 and W311 are essential residues and residue 309 has to be hydrophobic and possess an aromatic side chain, such as tryptophan or phenylalanine. When residue 308, which lies between tryptophans (W307 and W309) is changed from an acidic to a basic amino acid, the cytotoxic activity of the mutant is reduced to less than that of the wild type. It was shown by a toxin overlay assay that the cytotoxic activity of each mutant toxin correlates closely with affinity to GPI‐anchored proteins. These findings indicate that the WDW_W sequence in the tryptophan‐rich region plays an important role in the cytotoxic mechanism of alpha‐toxin, especially in the binding to GPI‐anchored proteins as cell receptors.     

Effects of phenylalanine substitutions in gramicidin A on the kinetics of channel formation in vesicles and channel structure in SDS micelles

The common occurrence of Trp residues at the aqueous-lipid interface region of transmembrane channels is thought to be indicative of its importance for insertion and stabilization of the channel in membranes. To further investigate the effects of Trp-->Phe substitution on the structure and function of the gramicidin channel, four analogs of gramicidin A have been synthesized in which the tryptophan residues at positions 9, 11, 13, and 15 are sequentially replaced with phenylalanine. The three-dimensional structure of each viable analog has been determined using a combination of two-dimensional NMR techniques and distance geometry-simulated annealing structure calculations. These phenylalanine analogs adopt a homodimer motif, consisting of two beta6.3 helices joined by six hydrogen bonds at their NH2-termini. The replacement of the tryptophan residues does not have a significant effect on the backbone structure of the channels when compared to native gramicidin A, and only small effects are seen on side-chain conformations. Single-channel conductance measurements have shown that the conductance and lifetime of the channels are significantly affected by the replacement of the tryptophan residues (Wallace, 2000; Becker et al., 1991). The variation in conductance appears to be caused by the sequential removal of a tryptophan dipole, thereby removing the ion-dipole interaction at the channel entrance and at the ion binding site. Channel lifetime variations appear to be related to changing side chain-lipid interactions. This is supported by data relating to transport and incorporation kinetics.     

Conformationally biased analogs of oxytocin.

Four diastereomeric analogs of oxytocin containing substituted phenylalanine in position 2 were synthesized. This modified phenylalanine side chain contained one methyl group attached to the beta-carbon and the second one at the 2' position of the aromatic ring. All analogs were found to be inhibitors of uterotonic activity of oxytocin with pA2 values ranging from 6.0 to 8.3; the most potent one (pA2 = 8.3) contained dimethylphenylalanine of the D-erythro configuration.     

Hydrophobicity of amino acid residues: differential scanning calorimetry and synthesis of the aromatic analogues of the polypentapeptide of elastin.

Relative hydrophobicities of aromatic amino acid residues are investigated by using differential scanning calorimetry (DSC) on 10 synthetic copolypentapeptides of poly(VPGVG) of elastin. Utilizing the hydrophobic-driven process of the inverse temperature transition exhibited by these polypentapeptides in aqueous solution, the relative hydrophobicities of Phe, Trp, and Tyr residues are determined by the critical temperature and heat of the transition. The DSC data for the aromatic residue containing copolypentapeptide aqueous solution indicate that tryptophan is the most hydrophobic amino acid residue, phenylalanine the third most hydrophobic on basis of transition temperature and the second on basis of transition heat. For tyrosine, significant differences are observed between the phenolic and the phenoxide anionic states. At pH 7, where tyrosine is protonated, it is found to be the second most hydrophobic amino acid residue on the basis of the transition temperature, whereas on the basis of the heat of transition, it is less hydrophobic than both tryptophan and phenylalanine. Changing the pH from pH 7 to pH 12, for example, for poly[0.8(VPGVG), 0.2(VPGYG)] in aqueous solution shifts the transition temperature from 7 to 49 degrees C with a dramatically reduced heat. On the basis of both the transition temperature scale and the heat of transition, the hydroxylated tyrosine appears less hydrophobic than glycine.     

Thermal Degradation of Aromatic Amino Acids

Aromatic amino acids (phenylalanine, tyrosine and tryptophan) were heated at 300°C under nitrogen and volatile compounds generated were examined. Twelve compounds in which many of them have aromatic rings were identified in the volatiles from thermal degradation of phenylalanine. Tyrosine and tryptophan produced some phenols and indoles, respectively, besides several compounds. Formation mechanisms of some compounds were also discussed.     

Comparison of the inhibitory effects of diverse amino acids and amino acid analogs on 12-O-tetradecanoylphorbol-13-acetate-induced ornithine decarboxylase activity in isolated epidermal cells

At a concentration of 1.25 mM, 14 amino acids were capable of inhibiting the induction of ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activity by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in isolated epidermal cells. The greatest percentages of inhibition of TPA-induced epidermal ornithine decarboxylase activity were as follows: cysteine, 98%; tryptophan, 74%; methionine, 64%; phenylalanine, 51%; glycine, 44%; asparagine, 43%; glutamic acid, 42%; leucine, 40%; and arginine, 39%. These amino acid treatments did not alter the time- and concentration-response curves for induction of ornithine decarboxylase activity by TPA. Moreover, there was no difference between the rates at which [3H]arginine, [3H]leucine, [3H]phenylalanine, [3H]methionine, [3H]tryptophan and [14C]cysteine were taken up by freshly isolated epidermal cells or incorporated into epidermal proteins. Arginine, phenylalanine and methionine inhibited the induction of ornithine decarboxylase activity by the tumor promoter to degrees comparable to those elicited by their analogs canavanine and homoarginine, beta-2-thienyl-DL-alanine, and ethionine, respectively. These amino acids and amino acid analogs did not alter the overall rate of protein synthesis. In contrast, both the amino acids and their analogs increased the rates of proteolysis in isolated epidermal cells, an effect which correlated well with the abilities of these different compounds to inhibit TPA-induced ornithine decarboxylase activity. Moreover, both methionine and phenylalanine decreased the half-life and increased the rate of heat denaturation of the TPA-induced enzyme, a result identical to that obtained after treatment with the analogs ethionine and beta-2-thienyl-DL-alanine, respectively. Taken together, these results suggest that millimolar concentrations of exogenous amino acids might induce the synthesis of abnormal proteins and nonfunctional enzymes. Therefore, it is speculated that the uptake of unbalanced amounts of amino acids into the epidermal target cells might alter the stability and the ultrastructure of the TPA-stimulated enzyme just as the amino acid analogs do.     

Weakly hydrated surfaces and the binding interactions of small biological solutes

Extended planar hydrophobic surfaces, such as are found in the side chains of the amino acids histidine, phenylalanine, tyrosine, and tryptophan, exhibit an affinity for the weakly hydrated faces of glucopyranose. In addition, molecular species such as these, including indole, caffeine, and imidazole, exhibit a weak tendency to pair together by hydrophobic stacking in aqueous solution. These interactions can be partially understood in terms of recent models for the hydration of extended hydrophobic faces and should provide insight into the architecture of sugar-binding sites in proteins.     

CoMFA and CoMSIA 3D-QSAR analysis of diaryloxy-methano-phenanthrene derivatives as anti-tubercular agents

Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) based on three-dimensional quantitative structure-activity relationship (3D-QSAR) studies were conducted on a series (44 compounds) of diaryloxy-methano-phenanthrene derivatives as potent antitubercular agents. The best predictions were obtained with a CoMFA standard model (q (2)=0.625, r (2)=0.994) and with CoMSIA combined steric, electrostatic, and hydrophobic fields (q (2)=0.486, r (2)=0.986). Both models were validated by a test set of seven compounds and gave satisfactory predictive r (2) values of 0.999 and 0.745, respectively. CoMFA and CoMSIA contour maps were used to analyze the structural features of the ligands to account for the activity in terms of positively contributing physicochemical properties: steric, electrostatic, and hydrophobic fields. The information obtained from CoMFA and CoMSIA 3-D contour maps can be used for further design of phenanthrene-based analogs as anti-TB agents. The resulting contour maps, produced by the best CoMFA and CoMSIA models, were used to identify the structural features relevant to the biological activity in this series of analogs. Further analysis of these interaction-field contour maps also showed a high level of internal consistency. This study suggests that introduction of bulky and highly electronegative groups on the basic amino side chain along with decreasing steric bulk and electronegativity on the phenanthrene ring might be suitable for designing better antitubercular agents.     

The characteristics of amino acid catabolism in bacteria of the genus Citrobacter

The comparison of the occurrence of enzymes effecting the deamination of dicarbon, aromatic and oxyamino acids, as well as transamination enzymes, in Citrobacter bacteria and the activity levels of these enzymes was made. The constant sign of such bacteria was the presence of serine and threonine dehydratase activity. 92% of the strains showed the presence of phenylalanine deaminase. No tryptophan activity was established. 96-98% of Citrobacter strains possessed phenylalanine, tyrosine and tryptophan aminotransferases with alpha-ketoglutaric acid functioning as the acceptor of the amino group.     

Crystal structures of glutamine:phenylpyruvate aminotransferase from Thermus thermophilus HB8: induced fit and substrate recognition

The following three-dimensional structures of three forms of glutamine:phenylpyruvate aminotransferase from Thermus thermophilus HB8 have been determined and represent the first x-ray analysis of the enzyme: the unliganded pyridoxal 5'-phosphate form at 1.9 A resolution and two complexes with 3-phenylpropionate and alpha-keto-gamma-methylthiobutyrate at 2.35 and 2.6 A resolution, respectively. The enzyme shows high activity toward phenylalanine, tyrosine, tryptophan, kynurenine, methionine, and glutamine. The enzyme is a homodimer, and each subunit is divided into an N-terminal arm and small and large domains. Based on its folding, the enzyme belongs to fold type I, aminotransferase subclass Ib. The subclass I aminotransferases whose structures have so far been determined exhibit a large movement of the small domain region upon binding of a substrate. Similarly, the glutamine:phenylpyruvate aminotransferase undergoes a large movement in part of the small domain to close the active site. The active-site pocket has a shape and size suitable to enclose the side chain of an aromatic amino acid or that of methionine. The inner side of the pocket is mostly hydrophobic, but also has polar sites. The kynurenine complex generated by computer modeling fits the pocket of the enzyme and its hydrophilic groups interact with the polar sites of the pocket.     

Collagen’s primary structure determines collagen:HSP47 complex stoichiometry

Collagens play important roles in development and homeostasis in most higher organisms. In order to function, collagens require the specific chaperone HSP47 for proper folding and secretion. HSP47 is known to bind to the collagen triple helix, but the exact positions and numbers of binding sites are not clear. Here, we employed a collagen II peptide library to characterize high-affinity binding sites for HSP47. We show that many previously predicted binding sites have very low affinities due to the presence of a negatively charged amino acid in the binding motif. In contrast, large hydrophobic amino acids such as phenylalanine at certain positions in the collagen sequence increase binding strength. For further characterization, we determined two crystal structures of HSP47 bound to peptides containing phenylalanine or leucine. These structures deviate significantly from previously published ones in which different collagen sequences were used. They reveal local conformational rearrangements of HSP47 at the binding site to accommodate the large hydrophobic side chain from the middle strand of the collagen triple helix and, most surprisingly, possess an altered binding stoichiometry in the form of a 1:1 complex. This altered stoichiometry is explained by steric collisions with the second HSP47 molecule present in all structures determined thus far caused by the newly introduced large hydrophobic residue placed on the trailing strand. This exemplifies the importance of considering all three sites of homotrimeric collagen as independent interaction surfaces and may provide insight into the formation of higher oligomeric complexes at promiscuous collagen-binding sites.     

A single surface tryptophan in the chitin-binding domain from Bacillus circulans chitinase A1 plays a pivotal role in binding chitin and can be modified to create an elutable affinity tag

Site-directed mutagenesis was carried out to investigate the roles of a number of highly conserved residues of the chitin-binding domain (ChBD) of Bacillus circulans chitinase A1 (ChiA1) in the binding of chitin. Analysis of single alanine replacement mutants showed that mutation of an exposed tryptophan residue (Trp(687)) impaired the binding to chitin, while mutation of other highly conserved residues, most carrying aromatic or hydrophobic side chains, did not significantly affect the binding activity. Interestingly, replacement of Trp(687) with phenylalanine significantly reduced chitin-binding activity at lower salt concentrations (0-1 M NaCl) but allowed strong binding to chitin at 2 M NaCl. Since Trp(687) is conserved among the ChBDs belonging to the bacterial ChiA1 subfamily, the data presented suggest a general mechanism in which this exposed tryptophan, which is located in the cleft formed between two beta-sheets as revealed by the solution structure [J. Biol. Chem. 275 (2000) 13654], makes a major contribution to ligand binding presumably through hydrophobic interactions. Furthermore, modulation of the chitin-binding activity by the conserved amino acid replacement (W687F) and a shift in the ionic strength of buffer has led to the development of an elutable affinity tag for single column purification of recombinant proteins.     

Transport of Aromatic Amino Acids by Pseudomonas aeruginosa

Kinetic studies of the transport of aromatic amino acids by Pseudomonas aeruginosa revealed the existence of two high-affinity transport systems which recognized the three aromatic amino acids. From competition data and studies on the exchange of preformed aromatic amino acid pools, the first transport system was found to be functional with phenylalanine, tyrosine, and tryptophan (in order of decreasing activity), whereas the second system was active with tryptophan, phenylalanine, and tyrosine. The two systems also transported a number of aromatic amino acid analogues but not other amino acids. Mutants defective in each of the two and in both transport systems were isolated and described. When the amino acids were added at low external concentrations to cells growing logarithmically in glucose minimal medium, the tryptophan pool very quickly became saturated. Under identical conditions, phenylalanine and tyrosine each accumulated in the intracellular pool of P. aeruginosa at a concentration which was 10 times greater than that of tryptophan.