Ribosomal binding and dipeptide formation by misacylated tRNA(Phe),S

Eight structurally modified peptidyl-tRNA(Phe),s were employed to study P-site binding and peptide bond formation in a cell-free system involving Escherichia coli ribosomes programmed with poly(uridylic acid). It was found that the two analogues (N-acetyl-D-phenylalanyl-tRNA(Phe) and N-acetyl-D-tyrosyl-tRNA(Phe] containing D-amino acids functioned poorly as donors in the peptidyltransferase reaction and that two N-acetyl-L-phenylalanyl-tRNA(Phe)'s differing from the prototype substrate in that they contained 2'- or 3'-deoxyadenosine at the 3'-terminus failed to form dipeptide at all when L-phenylalanyl-tRNA(Phe) was the acceptor tRNA. Interestingly, all four of these peptidyl-tRNA's bound to ribosomes to about the same extent as tRNA's that functioned normally as donors in the peptidyltransferase reaction, at least in the absence of competing peptidyl-tRNA species. Two peptidyl-tRNA's lacking an amino group were also tested. In comparison with N-acetyl-L-phenylalanyl-tRNA(Phe) it was found that trans-cinnamyl-tRNA(Phe) and 3-phenylpropionyl-tRNA(Phe)'s formed dipeptides to the extent of 53 and 80%, respectively, when L-phenylalanyl-tRNA(Phe)was used as the acceptor tRNA. N-Acetyl-beta-phenylalanyl-tRNA(Phe) was found to be the most efficient donor substrate studied. Both isomers transferred N-acetyl-beta-phenylalanine to L-phenylalanyl-tRNA(Phe); the nature of the dipeptides formed in each case was verified by HPLC in comparison with authentic synthetic samples. Further, the rate and extent of peptide bond formation in each case exceeded that observed with the control tRNA, N-acetyl-L-phenylalanyl-tRNA(Phe).     

Molecular insights into protein synthesis with proline residues

Proline is an amino acid with a unique cyclic structure that facilitates the folding of many proteins, but also impedes the rate of peptide bond formation by the ribosome. As a ribosome substrate, proline reacts markedly slower when compared with other amino acids both as a donor and as an acceptor of the nascent peptide. Furthermore, synthesis of peptides with consecutive proline residues triggers ribosome stalling. Here, we report crystal structures of the eukaryotic ribosome bound to analogs of mono‐ and diprolyl‐tRNAs. These structures provide a high‐resolution insight into unique properties of proline as a ribosome substrate. They show that the cyclic structure of proline residue prevents proline positioning in the amino acid binding pocket and affects the nascent peptide chain position in the ribosomal peptide exit tunnel. These observations extend current knowledge of the protein synthesis mechanism. They also revise an old dogma that amino acids bind the ribosomal active site in a uniform way by showing that proline has a binding mode distinct from other amino acids.     

Structural basis for the inability of chloramphenicol to inhibit peptide bond formation in the presence of A-site glycine

Ribosome serves as a universal molecular machine capable of synthesis of all the proteins in a cell. Small-molecule inhibitors, such as ribosome-targeting antibiotics, can compromise the catalytic versatility of the ribosome in a context-dependent fashion, preventing transpeptidation only between particular combinations of substrates. Classic peptidyl transferase center inhibitor chloramphenicol (CHL) fails to inhibit transpeptidation reaction when the incoming A site acceptor substrate is glycine, and the molecular basis for this phenomenon is unknown. Here, we present a set of high-resolution X-ray crystal structures that explain why CHL is unable to inhibit peptide bond formation between the incoming glycyl-tRNA and a nascent peptide that otherwise is conducive to the drug action. Our structures reveal that fully accommodated glycine residue can co-exist in the A site with the ribosome-bound CHL. Moreover, binding of CHL to a ribosome complex carrying glycyl-tRNA does not affect the positions of the reacting substrates, leaving the peptide bond formation reaction unperturbed. These data exemplify how small-molecule inhibitors can reshape the A-site amino acid binding pocket rendering it permissive only for specific amino acid residues and rejective for the other substrates extending our detailed understanding of the modes of action of ribosomal antibiotics.     

The energy utilized in protein breakdown by the ATP-dependent protease (La) from Escherichia coli

A crucial enzyme in the pathway for protein degradation in Escherichia coli is protease La, an ATP-hydrolyzing protease encoded by the lon gene. This enzyme degrades various proteins to small polypeptides containing 10-20 amino acid residues. To learn more about its energy requirement, we determined the number of ATP molecules hydrolyzed by the purified protease for each peptide bond cleaved. The enzyme hydrolyzed about 2 molecules of ATP for each new amino group generated with casein, bovine serum albumin, glucagon, or guanidinated casein as substrates, even though these proteins differ up to 20-fold in size and 3-4 fold in rates of hydrolysis of peptide bonds. Similar values for the stoichiometry (from 1.9 to 2.4) were obtained using fluorescamine or 2,4,6-trinitrobenzene sulfonic acid to estimate the appearance of new amino groups. These values appeared lower at 1 mM than at 10 mM Mg2+. The coupling between ATP and peptide bond hydrolysis appeared very tight. However, when the protease was assayed under suboptimal conditions (e.g. at lower pH or with ADP present), many more ATP molecules (from 3.5 to 12) were consumed per peptide bond cleaved. Our data would indicate that the early steps in protein degradation consume almost as much energy (2 ATPs for each cleavage) as does the formation of peptide bonds during protein synthesis.     

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.     

Protease-catalyzed fragment condensation via substrate mimetic strategy: a useful combination of solid-phase peptide synthesis with enzymatic methods.

The concept of substrate mimetic strategy represents a new powerful method in the field of enzymatic peptide synthesis. This strategy takes advantage of the shift in the site-specific amino acid moiety from the acyl residue to the ester-leaving group of the carboxyl component enabling acylation of the enzyme by nonspecific acyl residues. As a result, peptide bond formation occurs independently of the primary specificity of proteases. Moreover, because of the coupling of nonspecific acyl residues, the newly formed peptide bond is not subject to secondary hydrolysis achieving irreversible peptide synthesis. Here, we report the combination of solid-phase peptide synthesis with substrate mimetic-mediated enzymatic peptide fragment condensations. First, the utility of the oxime resin strategy for the synthesis of peptide fragments in the form of substrate mimetics esterified as 4-guanidinophenyl-, phenyl- and mercaptopropionic acid esters was investigated. The study was completed by using the resulting N(alpha)-protected peptide esters as acyl donors in trypsin-, alpha-chymotrypsin- and V8 protease-catalyzed fragment condensations.     

Peptide synthesis in aqueous-organic solvent mixtures with alpha-chymotrypsin immobilized to tresyl chloride-activated agarose

alpha-Chymotrypsin was immobilized with a high coupling yield (up to 80%) to tresyl chloride activated Sepharose CL-4B.The immobilized enzyme was tested for its ability to synthesize soluble peptides from N-acetylated amino acid esters as acyl donors and amino acid amides as acceptor amines in water-water-miscible organic solvent mixtures. It was found that the yield of peptide increased with increasing concentration of organic cosolvent. Almost complete synthesis (97%) of Ac-Phe-Ala-NH(2) was obtained from Ac-Phe-OMe using a sixfold excess of Ala-NH(2). The rate of peptide formation in aqueous-organic solvent mixtures was good. Thus, 0.1M peptide was formed in less than 2 h in 50 vol% DMF with 0.1 mg immobilized chymotrypsin/mL reaction mixture. The immobilized enzyme distinguished between the L and D configurations of acceptor amino acid amides even in high concentration of nonaqueous component (90% 1,4-butanediol). The effect of temperature was studied. It was found that both the yield of peptide and the stability of immobilized enzyme increased when the temperature was lowered. Experiments could be performed at subzero temperatures in the aqueous-organic solvent mixtures resulting in very high yield of peptide. After three weeks continuous operation at 4 degrees C in 50% DMF, the immobilized enzyme retained 66%of its original synthetic activity. The activity of the immobilized enzyme was better conserved with a preparation made from agarose with a higher tresyl group content compared to a preparation made from a lower activated agarose, indicating that multiple point of attachment has a favorable effect on the stability of the enzyme in aqueous-organic solvent mixtures. The major advantage of using water-miscible instead of water-immiscible organic solvents to promote peptide syntheses appears to be the increased solubility of substrates and products, making continuous operation possible.     

Hydrogen bonds in crystal structures of amino acids, peptides and related molecules

The results of a survey of 439 hydrogen bonds in 95 recently determined crystal structures of amino acids, peptides and related molecules suggest that the following generalizations hold true for linear (angle X-H---Y greater than 150 degrees) hydrogen bonds. (1) The charge on the acceptor group does not influence the length of a hydrogen bond. (2) For a given acceptor group, the hydrogen bond lengths increase in the order imidazolium N--H less than ammonium N-H less than guanidinium N-H; this order holds true for oxygen anion acceptor groups. Cl-ions and the uncharged oxygen of water molecules. (3) The uncharged imidazole N-H group forms shorter hydrogen than the amide N-H GROUP. (4) The carboxyl O-H groups form shorter hydrogen bonds than other hydroxyl groups. (5) The hydrogen bonds involving a halogen ion are longer than hydrogen bonds with other acceptors when corrected for their longer van der Walls radii. The observed differences between the lengths of hydrogen bonds formed by different donor and acceptor groups in amino acids and peptides, imply differences in the energetics of their formation.     

Peptide synthesis catalyzed by polyethylene glycol-modified chymotrypsin in organic solvents

Chymotrypsin modified with polyethylene glycol was successfully used for peptide synthesis in organic solvents. The benzene-soluble modified enzyme readily catalyzed both aminolysis of N-benzoyl-L-tyrosine p-nitroanilide and synthesis of N-benzoyl-L-tyrosine butylamide in the presence of trace amounts of water. A quantitative reaction was obtained when either hydrophobic or bulky amides of L- as well as D-amino acids were used as acceptor nucleophiles, while almost no reaction occurred with free amino acids or ester derivatives. The acceptor nucleophile specificity of modified chymotrypsin as a catalyst in the formation of both amide and peptide bonds in organic solvents was quite comparable to that in aqueous solution as well as to that of the leaving group in hydrolysis reactions. By contrast, the substrate specificity of modified chymotrypsin in organic solvents was different from that in water since arginine and lysine esters were found to be as effective as aromatic amino acids to form the acyl-enzyme with subsequent synthesis of a peptide bond.     

Conformational analysis of N-(tert.-amyloxycarbony-L-proline in the solid state and in solution.

The solid-state conformational analysis of t-AOC-L-Pro-OH has indicated that the molecules are not folded up to form an oxy-C7 peptide conformation, but rather that they are held together through intermolecular O-H .... 0 = C (urethane) hydrogen bonds. The tertiary amide bond is in the cis configuration. In solvents of high polarity strongly solvated species largely predominate. In cyclohexane solution non-associated and associated (involving the carboxyl C = O as the proton acceptor) species are simultaneously present. Obviously, the extent of association increases with increasing solute concentration. The amount of the oxy-C7 form, if any, should be extremely small. It is also demonstrated that CD measurements alone can lead to an incorrect picture of the conformational preferences of amino acid derivatives and small peptides in solution.     

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     

Amino acid–dependent stability of the acyl linkage in aminoacyl-tRNA

Aminoacyl-tRNAs are the biologically active substrates for peptide bond formation in protein synthesis. The stability of the acyl linkage in each aminoacyl-tRNA, formed through an ester bond that connects the amino acid carboxyl group with the tRNA terminal 3′-OH group, is thus important. While the ester linkage is the same for all aminoacyl-tRNAs, the stability of each is not well characterized, thus limiting insight into the fundamental process of peptide bond formation. Here, we show, by analysis of the half-lives of 12 of the 22 natural aminoacyl-tRNAs used in peptide bond formation, that the stability of the acyl linkage is effectively determined only by the chemical nature of the amino acid side chain. Even the chirality of the side chain exhibits little influence. Proline confers the lowest stability to the linkage, while isoleucine and valine confer the highest, whereas the nucleotide sequence in the tRNA provides negligible contribution to the stability. We find that, among the variables tested, the protein translation factor EF-Tu is the only one that can protect a weak acyl linkage from hydrolysis. These results suggest that each amino acid plays an active role in determining its own stability in the acyl linkage to tRNA, but that EF-Tu overrides this individuality and protects the acyl linkage stability for protein synthesis on the ribosome.     

Reaction mechanism, specificity and pH-dependence of peptide synthesis catalyzed by the metalloproteinase thermolysin

The initial rates of peptide bond formation catalyzed by the metalloproteinase thermolysin were determined. The dependence of the formation rates on the concentration of the carboxyl donor and the acceptor can be explained by a rapid-equilibrium random bireactant mechanism, in which the binding of one substrate has a positive influence on the binding of the other (synergism). The specificity of the enzyme for the donor and acceptor in the condensation reaction was further investigated by determining the apparent kinetic parameters kcat and Km for various substrates. The pH-dependence of the initial rates of synthesis was found to be identical to the pH-dependence of the hydrolytic action of the enzyme. The rates are also shown to be independent of the pKa of the amino group of the acceptor, indicating that deprotonation of the attacking nucleophile in the synthetic reaction is not rate-limiting.     

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.     

Epsilon-poly-L-lysine dispersity is controlled by a highly unusual nonribosomal peptide synthetase

Epsilon-Poly-L-lysine (epsilon-PL) consists of 25-35 L-lysine residues in isopeptide linkages and is one of only two amino acid homopolymers known in nature. Elucidating the biosynthetic mechanism of epsilon-PL should open new avenues for creating novel classes of biopolymers. Here we report the purification of an epsilon-PL synthetase (Pls; 130 kDa) and the cloning of its gene from an epsilon-PL-producing strain of Streptomyces albulus. Pls was found to be a membrane protein with adenylation and thiolation domains characteristic of the nonribosomal peptide synthetases (NRPSs). It had no traditional condensation or thioesterase domain; instead, it had six transmembrane domains surrounding three tandem soluble domains. These tandem domains iteratively catalyzed L-lysine polymerization using free L-lysine polymer (or monomer in the initial reaction) as acceptor and Pls-bound L-lysine as donor, directly yielding chains of diverse length. Thus, Pls is a new single-module NRPS having an amino acid ligase-like catalytic activity for peptide bond formation.     

Resin probe analysis. II. Amino acid coupling reactions.

Resin probe analysis has been employed to evaluate the availability of dicyclohexylcarbodiimide (DCC)-activated amino acids, the relationship between coupling time and reaction yield, and the influence of triethylamine (TEA) concentration on peptide bond formation. Results are presented for five amino acids which indicate that the coupling reactions plateau within 5 min, and no significant increase in yield is observed for longer incubation times. Large decreases in coupling yield (70–90%) were observed at concentrations of TEA above 0.01 m. Inactivation appears to be dependent in part upon amino acid structural features. In the absence of TEA, DCC-activated t-butyloxycarbonyl (Boc)-glycine was stable in the activated state for hours. peptide bond formation showed little or no amino acid concentration-dependence in the range of 0.01–0.04 m. Resin probe experiments provide quantitative data on reaction progress and factors that influence the availability and reactivity of activated amino acids.     

Structural-functional organization of protein hormone molecules

Our experimental results and literature data based on present-day concepts of molecular evolution of proteins have formed the basis for a hypothesis on the structural-functional organization of protein hormone molecules. This hypothesis postulates the existence of three types of the peptide chain sites, which play different roles in the biological action of hormones, i.e. effector (active), acceptor (binding) and accessory ones. The local similarity spectra have been computed and used for a comparison of the amino acid sequences of related hormones belonging to different groups. The positions of maxima, slopes and minima in these spectra of the molecules of peptide and some protein hormones are correlated with a most probable localization of the effector, acceptor and accessory sites, respectively.     

The structural basis for specificity in human ABO(H) blood group biosynthesis

The human ABO(H) blood group antigens are produced by specific glycosyltransferase enzymes. An N-acetylgalactosaminyltransferase (GTA) uses a UDP-GalNAc donor to convert the H-antigen acceptor to the A antigen, whereas a galactosyltransferase (GTB) uses a UDP-galactose donor to convert the H-antigen acceptor to the B antigen. GTA and GTB differ only in the identity of four critical amino acid residues. Crystal structures at 1.8-1.32 A resolution of the GTA and GTB enzymes both free and in complex with disaccharide H-antigen acceptor and UDP reveal the basis for donor and acceptor specificity and show that only two of the critical amino acid residues are positioned to contact donor or acceptor substrates. Given the need for stringent stereo- and regioselectivity in this biosynthesis, these structures further demonstrate that the ability of the two enzymes to distinguish between the A and B donors is largely determined by a single amino acid residue.     

Evidence for RNA in the peptidyl transferase center of Escherichia coli ribosomes as indicated by fluorescence.

A coumarin derivative was covalently attached to either the amino acid or the 5' end of phenylalanine-specific transfer RNA (tRNA(phe)). Its fluorescence was quenched by methyl viologen when the tRNA was free in solution or bound to Escherichia coli ribosomes. Methyl viologen as a cation in solution has a strong affinity for the ionized phosphates of a nucleic acid and so can be used to qualitatively measure the presence of RNA in the immediate vicinity of the tRNA-linked coumarins upon binding to ribosomes. Fluorescence lifetime measurements indicate that the increase in fluorescence quenching observed when the tRNAs are bound into the peptidyl site of ribosomes is due to static quenching by methyl viologen bound to RNA in the immediate vicinity of the fluorophore. The data lead to the conclusion that the ribosome peptidyl transferase center is rich in ribosomal RNA. Movement of the fluorophore at the N-terminus of the nascent peptide as it is extended or movement of the tRNA acceptor stem away from the peptidyl transferase center during peptide bond formation appears to result in movement of the probe into a region containing less rRNA.     

Peptidyl-prolyl-tRNA at the ribosomal P-site reacts poorly with puromycin

Despite remarkable recent progress in our chemical and structural understanding of the mechanisms of peptide bond formation by the ribosome, only very limited information is available about whether amino acid side chains affect the rate of peptide bond formation. Here, we generated a series of peptidyl-tRNAs that end with different tRNA-attached amino acids in the P-site of the Escherichia coli ribosome and compared their reactivity with puromycin, a rapidly A-site-accessing analog of aminoacyl-tRNAs. Among the 20 amino acids examined, proline was found to receive exceptionally slow peptidyl transfer to puromycin. These results raise a possibility that the peptidyl transferase activity of the ribosome may have some specificity with regard to the P-site amino acids.