Selenium toxicity: aminoacylation and Peptide bond formation with selenomethionine

Selenomethionine and methionine were compared as substrates for in vitro aminoacylation, ribosome binding, and peptide bond formation with preparations from wheat germ. Selenomethionine paralleled methionine in all steps of the translation process except peptide bond formation. Peptide bond formation with the initiating species of tRNA^Met demonstrated that selenomethionyl-tRNA^Met was less effective as a substrate than was methionyl-tRNA_f^Met. Participation of selenomethionine in the initiation process of translation could be expected to reduce the overall rate of protein synthesis and might aid in explaining selenium toxicity in selenium-sensitive plants.     

Phylogenetic analysis of condensation domains in NRPS sheds light on their functional evolution

Background  

Non-ribosomal peptide synthetases (NRPSs) are large multimodular enzymes that synthesize a wide range of biologically active natural peptide compounds, of which many are pharmacologically important. Peptide bond formation is catalyzed by the Condensation (C) domain. Various functional subtypes of the C domain exist: An^LC_L domain catalyzes a peptide bond between two L-amino acids, a^DC_L domain links an L-amino acid to a growing peptide ending with a D-amino acid, a Starter C domain (first denominated and classified as a separate subtype here) acylates the first amino acid with a β -hydroxy-carboxylic acid (typically a β -hydroxyl fatty acid), and Heterocyclization (Cyc) domains catalyze both peptide bond formation and subsequent cyclization of cysteine, serine or threonine residues. The homologous Epimerization (E) domain flips the chirality of the last amino acid in the growing peptide; Dual E/C domains catalyze both epimerization and condensation.     

The Role of Initiator tRNAi met in Fidelity of Initiation of Protein Synthesis

The proper arrangement of amino acids in a protein determines its proper function, which is vital for the cellular metabolism. This indicates that the process of peptide bond formation requires high fidelity. One of the most important processes for this fidelity is kinetic proofreading. As biochemical experiments suggest that kinetic proofreading plays a major role in ensuring the fidelity of protein synthesis, it is not certain whether or not a misacylated tRNA would be corrected by kinetic proofreading during the peptide bond formation. Using 2-layered ONIOM (QM/MM) computational calculations, we studied the behavior of misacylated tRNAs and compared the results with these for cognate aminoacyl-tRNAs during the process of peptide bond formation to investigate the effect of nonnative amino acids on tRNAs. The difference between the behavior of initiator tRNA_i ^met compared to the one for the elongator tRNAs indicates that only the initiator tRNA_i ^met specifies the amino acid side chain.     

Cathepsin D generates gamma-endorphin from beta-endorphin.

A crude extract of porcine anterior pituitary was found to contain endopeptidase activity that splits the Leu⁷⁷-Phe⁷⁸ peptide bond of β-lipotropin in a pH range 3.0–7.0. The specificity and susceptibility to pepstatin of pituitary extract were the same as those of cathepsin D (E.C. 3.4.23.5) isolated from calf brain by affinity chromatography. Cathepsin D was shown to split the same Leu-Phe peptide bond of β-endorphin, leading to the formation of γ-endorphin. Based on the above data it is suggested that cathepsin D is a major factor in the generation of endorphins of intermediate size.     

Energetics of peptide bond formation at elevated temperatures

Summary The free energies of formation of the peptide bond between carbobenzoxy-glycine and L-phenylalanine amide in aqueous solution at temperatures up to 60°C were calculated from experimentally determined equilibrium constants. The reaction was catalyzed by a thermophylic enzyme. The thermodynamic energy barrier to peptide bond formation was found to decrease with increasing temperature: the standard free energy of peptide bond formation did appear to become negative in the region of 60°C. The possible significance of these results for peptide bond formation under prebiotic conditions is discussed.     

Characterization of a Reduced Form of Plasma Plasminogen as the Precursor for Angiostatin Formation

Plasma plasminogen is the precursor of the tumor angiogenesis inhibitor, angiostatin. Generation of angiostatin in blood involves activation of plasminogen to the serine protease plasmin and facilitated cleavage of two disulfide bonds and up to three peptide bonds in the kringle 5 domain of the protein. The mechanism of reduction of the two allosteric disulfides has been explored in this study. Using thiol-alkylating agents, mass spectrometry, and an assay for angiostatin formation, we show that the Cys⁴⁶²-Cys⁵⁴¹ disulfide bond is already cleaved in a fraction of plasma plasminogen and that this reduced plasminogen is the precursor for angiostatin formation. From the crystal structure of plasminogen, we propose that plasmin ligands such as phosphoglycerate kinase induce a conformational change in reduced kringle 5 that leads to attack by the Cys⁵⁴¹ thiolate anion on the Cys⁵³⁶ sulfur atom of the Cys⁵¹²-Cys⁵³⁶ disulfide bond, resulting in reduction of the bond by thiol/disulfide exchange. Cleavage of the Cys⁵¹²-Cys⁵³⁶ allosteric disulfide allows further conformational change and exposure of the peptide backbone to proteolysis and angiostatin release. The Cys⁴⁶²-Cys⁵⁴¹ and Cys⁵¹²-Cys⁵³⁶ disulfides have −/+RHHook and −LHHook configurations, respectively, which are two of the 20 different measures of the geometry of a disulfide bond. Analysis of the structures of the known allosteric disulfide bonds identified six other bonds that have these configurations, and they share some functional similarities with the plasminogen disulfides. This suggests that the −/+RHHook and −LHHook disulfides, along with the −RHStaple bond, are potential allosteric configurations.     

Microwave-assisted solid-phase peptide synthesis of the 60–110 domain of human pleiotrophin on 2-chlorotrityl resin

A fast and efficient microwave-assisted solid phase peptide synthesis (MW-SPPS) of a 51mer peptide, the main heparin-binding site (60–110) of human pleiotrophin (hPTN), using 2-chlorotrityl chloride resin (CLTR-Cl) following the 9-fluorenylmethyloxycarbonyl/tert-butyl (Fmoc/tBu) methodology and with the standard N,N′-diisopropylcarbodiimide/1-hydroxybenzotriazole (DIC/HOBt) coupling reagents, is described. An MW-SPPS protocol was for the first time successfully applied to the acid labile CLTR-Cl for the faster synthesis of long peptides (51mer peptide) and with an enhanced purity in comparison to conventional SPPS protocols. The synthesis of such long peptides is not trivial and it is generally achieved by recombinant techniques. The desired linear peptide was obtained in only 30 h of total processing time and in 51% crude yield, in which 60% was the purified product obtained with 99.4% purity. The synthesized peptide was purified by reversed phase high performance liquid chromatography (RP-HPLC) and identified by electrospray ionization mass spectrometry (ESI-MS). Then, the regioselective formation of the two disulfide bridges of hPTN 60–110 was successfully achieved by a two-step procedure, involving an oxidative folding step in dimethylsulfoxide (DMSO) to form the Cys⁷⁷–Cys¹⁰⁹ bond, followed by iodine oxidation to form the Cys⁶⁷–Cys⁹⁹ bond.     

Clinical Use of Aromatase Inhibitors: Current Data and Future Perspectives

Abstract

A systematic procedure for the kinetic study of irreversible inhibition when the enzyme is consumed in the reaction which it catalyses, has been developed and analysed. Whereas in most reactions the enzymes are regenerated after each catalytic event and serve as reusable transacting effectors, in the consumed enzymes each catalytic center participates only once and there is no enzyme turnover. A systematic kinetic analysis of irreversible inhibition of these enzyme reactions is presented. Based on the algebraic criteria proposed in this work, it should be possible to evaluate either the mechanism of inhibition (complexing or non-complexing), or the type of inhibition (competitive, non-competitive, uncompetitive, mixed non-competitive). In addition, all kinetic constants involved in each case could be calculated. An experimental application of this analysis is also presented, concerning peptide bond formation in vitro. Using the puromycin reaction, which is a model reaction for the study of peptide bond formation in vitro and which follows the same kinetic law as the enzymes under study, we have found that: (i) the antibiotic spiramycin inhibits the puromycin reaction as a competitive irreversible inhibitor in a one step mechanism with an association rate constant equal to 1.3 × 10⁴M^-1s^-1 and, (ii) hydroxylamine inhibits the same reaction as an irreversible non-competitive inhibitor also in a one step mechanism with a rate constant equal to 1.6 × 10^-3 M^-1s^-1.     

The role of metal ions in chemical evolution: Polymerization of alanine and glycine in a cation-exchanged clay environment

Summary The effect of the exchangeable cation on the condensation of glycine and alanine was investigated using a series of homoionic bentonites. A cycling procedure of drying, warming and wetting was employed. Peptide bond formation was observed, and the effectiveness of metal ions to catalyze the condensation was Cu²⁺ > Ni²⁺ Zn²⁺ > Na⁺. Glycine showed 6% of the monomer incorporated into oligomers with the largest detected being the pentamer. Alanine showed less peptide bond formation (a maximum of 2%) and only the dimer was observed.     

Influence of sample pH on the conformational backbone dynamics of a pseudotripeptide (H-Tyr-TicΨ[CH2-NH] Phe-OH) incorporating a reduced peptide bond: An NMR investigation

In the present paper we investigate the influence of sample pH on the conformational and dynamical properties of the pseudotripeptide H-Tyr-TicΨ[CH₂NH]Phe-OH(TIP[Ψ]:Tic: l, 2, 3, 4,-tetrahydroisoquinoline-3-carboxylic acid) using various one- and two-dimensional nmt techniques in conjunction with molecular modeling. Studies were conducted at three different pH levels-corresponding to the zwitterionic peptide containing a formal positive charge(pH 3. 1).the deprotonated molecule(pH 9. 1), and a situation at neutral pH(pH 7. 2) involving both protonated and deprotonated states of the reduced peptide bond. Analysis of the one-dimensional¹H-nmr spectra reveals that in solution TIP[Ψ]is in slow dynamic exchange between conformations containing cis and trans configurations of the Tyr-Tic bond. An nmr pH dependence study of the cis:trans ratio indicated that the exchange process was governed by the protonation state of the reduced bond amine. From the nmr data, reduced peptide bond pK_avalues of 6. 5 and 7. 5 were determined for the cis and trans conformers, respectively. It was concluded that conformations containing a trans Tyr-Tic bond are stabilized at law pH by an intramolecular hydrogen bond between the Tyr carbonyl and the reduced peptide bond protonated amine. This observation was corroborated by molecular mechanics investigations that revealed low energy trans structures compatible with nmr structural data, and furthermore, were consistently characterized by the existence of a strong N⁺ H?O? C interaction closing a seven-membered cycle. The dynamics of cis-trans isomerization about the Tyr-Tic peptide bond were probed by nmr exchange experiments. The selective presaturation of exchanging resonances carried out at several temperatures between 50 and 70°C allowed the determination of isomerization rate constants as well as thermodynamic activation parameters. ΔG^≠ values were in close agreement with the cis → trans energy barrier found in X-Pro peptide fragments (～83 kJ/mol).A large entropic barrier determined for the trans → cis conversion of TIP[Ψ](5. 7 JK^?1 mol^?1 at pH 3. 1; 6. 5 JK^?1 mol^?1 at pH 9. 1) is discussed in terms of decreased solvent molecular ordering around the conformers possessing a trans Tyr-Tic bond. Evidence that the neutral form of the reduced peptide bond gains rigidity upon protonation was obtained from relaxation measurements in the rotating frame. T_Jp measurements of several protons in the vicinity of the reduced peptide bond were made as a function of spin-lock field. Quantitative analysis of the relaxation data indicated that chemical shift fluctuations in the 10^?4-10^?5s range were more pronounced in the case of deprotonated TIP[Ψ]. Results of molecular dynamics simulations in addition to ³ J _αβ coupling constant measurements support the experimentally observed greater flexibility in the C-terminal region of TIP[Ψ]. © 1995 John Wiley & Sons, Inc.     

Hydroxamate-based colorimetric assay to assess amide bond formation by adenylation domain of nonribosomal peptide synthetases

We demonstrated the usefulness of a hydroxamate-based colorimetric assay for predicting amide bond formation (through an aminoacyl-AMP intermediate) by the adenylation domain of nonribosomal peptide synthetases. By using a typical adenylation domain of tyrocidine synthetase (involved in tyrocidine biosynthesis), we confirmed the correlation between the absorbance at 490 nm of the l-Trp–hydroxamate–Fe³⁺ complex and the formation of l-Trp–l-Pro, where l-Pro was used instead of hydroxylamine. Furthermore, this assay was adapted to the adenylation domains of surfactin synthetase (involved in surfactin biosynthesis) and bacitracin synthetase (involved in bacitracin biosynthesis). Consequently, the formation of various aminoacyl l-Pro formations was observed.     

Nmr studies of the molecular conformations in the linear oligopeptides H-(L-Ala)n-L-Pro-OH

The molecular conformations of the linear oligopeptides H-(L -Ala)_n-L -Pro-OH, with n = 1,2 and 3, have been investigated. ¹³C nmr observation of the equilibrium between the cis and trans forms of the Ala-Pro peptide bond indicated the occurrence of nonrandom conformations in solutions of these flexible peptides. The formation of the nonrandom species containing the cis form of the Ala-Pro bond was found to depend on the deprotonation of the carboxylic acid group of proline, the solvent, and the ionic strength in aqueous solution. The influence of intramolecular hydrogen bonding on the relative conformational energies of the species containing the cis and trans Ala-Pro peptide bond was studied by comparison of the peptides H-(Ala)_n-Pro-OH with analogous molecules where hydrogen bond formation was excluded by the covalent structure. In earlier work a hydrogen bond between the protonated terminal carboxylic acid group and the carbonyl oxygen of the penultimate amino acid residue had been suggested to stabilize conformations including trans proline. For the systems described here this hypothesis can be ruled out, since the cis:trans ratio is identical for molecules with methyl ester protected and free protonated terminal carboxylic acid groups of proline. Direct evidence for hydrogen bond formation between the deprotonated terminal carboxylic acid group and the amide proton of the penultimate amino acid residue in the molecular species containing cis proline was obtained from ¹H nmr studies. However, the cis:trans ratio of the Ala-Pro bond was not affected by N-methylation of the penultimate amino acid residue, which prevents formation of this hydrogen bond. Overall the experimental observations lead to the conclusion that the relative energies of the peptide conformations including cis or trans proline are mainly determined by intramolecular electrostatic interactions, whereas in the molecules considered, intramolecular hydrogen bonding is a consequence of specific peptide backbone conformations rather than a cause for the occurrence of energetically favored species. Independent support for this conclusion was obtained from model consideration which indicated that electrostatic interactions between the terminal carboxylic acid group and the carbonyl oxygen of the penultimate amino acid residue could indeed account for the observed relative conformational energies of the species containing cis and trans proline, respectively.     

Free energies and equilibria of peptide bond hydrolysis and formation

For every n amino acids linked in a protein there are n − 1 peptide bonds. The free energy of peptide bond hydrolysis and formation in aqueous solution defines the equilibrium position between peptide and amino acid hydrolysis products. Yet few experimental values exist. With a minimum of assumptions, this paper deduces the free energies of hydrolysis of a variety of peptide bonds. Formation of a dipeptide from two amino acids is about eight times more difficult than subsequent condensations of an amino acid to a dipeptide or longer chain. Condensation of an amino acid to a peptide of any size is five times more difficult than joining two smaller peptides of at least dipeptide size. Thus in an abiogenesis scenario there is a kind of nucleation in peptide bond formation with the initial condensation of two amino acids to yield a dipeptide more difficult than subsequent condensations to a growing chain. © 1998 John Wiley & Sons, Inc. Biopoly 45: 351–353, 1998     

Kinetics of the competitive reactions of isomerization and peptide bond cleavage at l‐α‐ and d‐β‐aspartyl residues in an αA‐crystallin fragment

d ‐β‐aspartyl (Asp) residue has been found in a living body such as aged lens crystallin, although l ‐α‐amino acids are constituents in natural proteins. Isomerization from l ‐α‐ to d ‐β‐Asp probably modulates structures to affect biochemical reactions. At Asp residue, isomerization and peptide bond cleavage compete with each other. To gain insight into how fast each reaction proceeds, the analysis requires the consideration of both pathways simultaneously and independently. No information has been provided, however, about these competitive processes because each reaction has been studied separately. The contribution of Asp isomers to the respective pathways has still been veiled. In this work, the two competitive reactions, isomerization and spontaneous peptide bond cleavage at Asp residue, were simultaneously observed and compared in an αA‐crystallin fragment, S⁵¹LFRTVLD⁵⁸SG⁶⁰ containing l ‐α‐ and d ‐β‐Asp58 isomers. The kinetics showed that the formation of l ‐ and d ‐succinimide (Suc) intermediate, as a first step of isomerization, was comparable at l ‐α‐ and d ‐β‐Asp. Although l ‐Suc was converted to l ‐β‐Asp, d ‐Suc was liable to return to the original d ‐β‐Asp, the reverse reaction marked enough to consider d ‐β‐Asp as apparently stable. d ‐β‐Asp was also resistant to the peptide bond cleavage. Such apparent less reactivity is probably the reason for gradual and abnormal accumulation of d ‐β‐Asp in a living body under physiological conditions. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

The active site of the ribosome is composed of two layers of conserved nucleotides with distinct roles in peptide bond formation and peptide release

Peptide bond formation and peptide release are catalyzed in the active site of the large subunit of the ribosome where universally conserved nucleotides surround the CCA ends of the peptidyl- and aminoacyl-tRNA substrates. Here, we describe the use of an affinity-tagging system for the purification of mutant ribosomes and analysis of four universally conserved nucleotides in the innermost layer of the active site: A2451, U2506, U2585, and A2602. While pre-steady-state kinetic analysis of the peptidyl transferase activity of the mutant ribosomes reveals substantially reduced rates of peptide bond formation using the minimal substrate puromycin, their rates of peptide bond formation are unaffected when the substrates are intact aminoacyl-tRNAs. These mutant ribosomes do, however, display substantial defects in peptide release. These results reveal a view of the catalytic center in which an inner shell of conserved nucleotides is pivotal for peptide release, while an outer shell is responsible for promoting peptide bond formation.     

How the imidazole ring modulates amyloid formation of islet amyloid polypeptide: A chemical modification study

BackgroundThe misfolding of human islet amyloid polypeptide (hIAPP) is an important pathological factor on the onset of type 2 diabetes. A number of studies have been focused on His¹⁸, the only histidine of hIAPP, whose imidazole ring and the protonation state might impact hIAPP amyloid formation, but the exact mechanism remains unclear.MethodsWe used diethylpyrocarbonate (DEPC) to specifically modify His¹⁸ and obtained mono-ethyloxyformylated hIAPP (DMI). Thioflavin T based fluorescence, transmission electronic microscopy, circular dichroism spectroscopy, fluorescence dye leakage, Fourier transform infrared spectroscopy and replica-exchange molecular dynamics (REMD) simulation were applied to study the impact of DEPC-modification on hIAPP amyloid formation.ResultsAfter an ethyl-acetate group was introduced to the His¹⁸ of hIAPP by diethylpyrocarbonate (DEPC) modification, the pH dependent hIAPP fibrillation went to the opposite order and the number of intra-molecular hydrogen bonds decreased, while the possibility of His¹⁸ participating in the formation of α-helical structures increased. Furthermore, the membrane–peptide interaction and ion–peptide interaction were both impaired.ConclusionsThe intramolecular hydrogen bond formation by His¹⁸ and the possibility of His¹⁸ participating in the formation of α-helical structures greatly modulated the manner of hIAPP amyloid formation. The imidazole ring directly participates in the hIAPP–membrane/ion interaction.General significanceDEPC modification is an alternative approach to investigate the role of the imidazole ring during amyloid formation.     

Role of GTP in the Positioning of Formylmethionyl-tRNA<Subscript>f</Subscript> on the <Emphasis Type="Italic">E. coli</Emphasis> Ribosome

FORMATION of the E. coli initiation complex between ribosomal subunits, natural messenger RNA and formyl-methionyl-tRNA_f (fMet-tRNA_f) requires the presence of initiation factors and GTP^1–3. In the binding reaction, GTP can be replaced by an analogue, guanylyl-5′-methylene-diphosphonate (GMP-PCP), but the complex does not then react with puro-mycin. Hydrolysis of GTP is therefore required for the formation of an active initiation complex able to carry out peptide bond formation⁴.     

Synthesis and conformational analysis of aib-containing peptide modelling for N-glycosylation site in N-glycoprotein

A tetrapetide containing an Aib residue, Boc-Asn-Aib-Thr-Aib-OMe, was synthesized as a peptide model for the N-glycosylation site in N-glycoproteins. Backbone conformation of the peptide and possible intramolecular interaction between the Asn and Thr side chains were elucidated by means of n.m.r. spectroscopy. Temperature dependence of NH proton chemical shift and NOE experiments showed that Boc-Asn-Aib-Thr-Aib-OMe has a tendency to form a β-turn structure with a hydrogen bond involving Thr and Aib⁴ NH groups. Incorporation of Aib residues in the peptide model promotes folding of the peptide backbone. With folded backbone conformation, carboxyamide protons of the Asn residue are not involved in hydrogen bond network, while the OH group of the Thr residue is a candidate for a hydrogen bond in DMSO-d₆ solution.     

Conformational characteristics of polypeptides containing isolated L-proline residues with cis peptide bonds

The conformational characteristics of the peptide sequence X-l-Pro, where X  Gly or l-Ala and the peptide bond joining X and l-Pro is cis, are evaluated. Semi-empirical potential functions are used to estimate the contributions to the conformational energy made by the non-bonded van der Waals' and electrostatic interactions and the intrinsic torsional potentials about the NC^a and C^aC′ bonds. Rotations φ₁ and ψ₁ about the NC^a and C^aC′ bonds in residue X and rotation ψ₂ about the C^aC′ bond in l-Pro are permitted, while the angle of rotation φ₂ about the NC^a bond in l-Pro is fixed at 120 ° by the pyrrolidine ring. The presence of the cis peptide bond connecting X and l-Pro renders the backbone rotations φ₁, ψ₁ in X dependent upon the rotation ψ₂ about the C^aC′ bond in l-Pro. (Interdependence of rotations in neighboring residues joined by a cis peptide bond was previously observed in l-alanine oligomers.) The number of energetically allowed conformations for the Gly and l-Ala residues preceding a cis peptide bond l-Pro residue are found to be substantially reduced from those permitted when the peptide bond is trans or when l-Pro is replaced by an amino acid residue. On the other hand, ψ₂ = 100 to 160 ° (cis′) and 300 to 0 ° (trans′) are found to be the lowest energy conformations of the l-Pro residue irrespective of the cis or trans conformation of the X-l-Pro peptide bond.