Catalytic effects of histidine enantiomers and glycine on the formation of dileucine and dimethionine in the salt-induced peptide formation reaction

The salt-induced peptide formation (SIPF) reaction takes place readily under mild reaction conditions and proceeds via a copper complex. Its ease of reaction and the universality for prebiotic scenarios add weights to the arguments in favour of the importance of peptide and proteins in the tug of war with the RNA world hypothesis. In addition, the SIPF reaction has a preference for l-form amino acids in dipeptide formation, casting light on the puzzle of biohomochirality, especially for the amino acids with aliphatic side chains. A detailed investigation on the behaviour of aliphatic leucine in the SIPF reaction is presented in this paper, including the catalytic effects of glycine, l- and d-histidine as well as the stereoselectivity under all the reaction conditions above. The results show a relatively low reactivity and stereoselectivity of leucine in the SIPF reaction, while both glycine and histidine enantiomers remarkably increase the yields of dileucine by factors up to 40. Moreover, a comparative study of the effectiveness of l- and d-histidine in catalysing the formation of dimethionine was also carried out and extends the scope of mutual catalysis by amino acid enantiomers in the SIPF reaction.     

Indications towards a stereoselectivity of the salt-induced peptide formation reaction

Different reaction yields for l- and d-alanine in the salt-induced peptide formation (SIPF) reaction, differences in the circular dichroism spectra and the complex formation constants of the involved chlorocuprate complexes point at a stereoselective differentiation between the two stereoisomers in the SIPF reaction and give a possible explanation towards the origin of homochirality in the process of the origin of life. An explanation of the observed effects can for the time being only be based on assumptions but could possibly be related to the inherent chirality of the Cu^II ion as a central atom of the [CuCl(gly)(glyH₂)(H₂O)₂]⁺ complex due to parity violation in weak interactions and to amplification of chirality related to the structural properties of the complex.     

Investigations on the mechanism of the salt-induced peptide formation

The applicability of the salt-induced peptide formation in aqueous solution — the simplest model so far for peptide synthesis under primitive earth conditions — is demonstrated for valine as another amino acid, and the formation of mixed peptides in systems containing glycine, alanine and valine is investigated. The dominant dipeptides formed are Gly-Gly, Gly-Ala and Gly-Val, at longer reaction times sequence inversion produces Ala-Gly and, considerably slower, Val-Gly. Ala-Ala is also produced and the relative amounts of the diastereomers prove the high conservation of optical purity of the original amino acids over a considerable time. The results lead to some further conclusions about the reaction mechanism and the possible dominance of peptide sequences in primordial dipeptides.     

Arginine:glycine amidinotransferase. A comparative study in lizard and mouse tissues

Kidney transamidinase activity in the lizard (Calotes versicolor), like that in the mouse, showed the pH optimum at 7.4. The lizard enzyme was inhibited to a greater degree than the mouse enzyme at high concentrations (greater than 20 mM) of L-arginine and glycine. Kidney and liver in the lizard and kidney and pancreas in the mouse were the tissues with high transamidinase activity. While transamidinase activity was widely distributed in mouse tissues, the enzyme was found to be restricted only to a few tissues in the lizard. Hydrocortisone administration into male lizards did not significantly alter the transamidinase levels in kidney and liver.     

The glycine cleavage system: Composition,reaction mechanism,and physiological significance

Summary The glycine cleavage system catalyzes the following reversible reaction: Glycine + THF + NAD⁺ 5,10-methylene-THF + + CO₂ + NH₃ + NADHReversibility of the overall reaction was established through the studies with the enzymes prepared from liver mitochondria of rat and cock and from extracts ofArthrobacter globiformis grown on glycine. The glycine cleavage system is composed from four protein components. The four proteins were revealed to exist originally as an enzyme complex in the liver mitochondria. Partial reactions of glycine cleavage and glycine synthesis were studied in detail with partially purified individual protein components. Particularly a protein-bound intermediate of glycine metabolism could be isolated and its nature and role were clarified. A tentative scheme was presented to explain the whole process of the reversible glycine cleavage.The glycine cleavage system was shown to represent the major pathway of catabolism of both glycine and serine in vertebrates, including mammals, birds, reptiles, amphibians, and fishes. Serine catabolism in these animals proceeds mainly by way of the cleavage of serine to form methylene-THF and glycine rather than deamination by serine dehydratase. In ureotelic and ammonotelic animals methylene-THF formed from the -carbon of glycine as well as the-carbon of serine could be further oxidized to CO₂ in either the mitochondria or the soluble tissue fractions, while in uricotelic animals methylene-THF could hardly be oxidized to CO₂ and instead, was utilized mostly for purine synthesis. Glycine synthesis by the glycine cleavage system did not appear to have appreciable physiological significance in animals.Abbreviation THF dl,l-tetrahydrofolic acid. an invited article     

Arginine and glycine stimulate creatine synthesis in creatine transporter 1-deficient lymphoblasts

Creatine transporter 1 (CT1) defect is an X-linked disease that causes severe neurological impairment. No treatment has been available for this condition so far. Because the transport of creatine (Cr) precursors Gly and Arg is not affected in this disorder, we tested the possible corrective effect of these two amino acids on Cr depletion in lymphoblasts lacking the transporter. Substrates enriched with Arg or Arg plus Gly increased the concentration of intracellular Cr in affected cells as well as in control cells. The greatest effect was obtained with 10 and 15 mM Arg and 10mM Arg plus Gly. These results encourage an in vivo trial with Cr precursors in CT1 defect.     

Arginine modification by phenylglyoxal and (p-hydroxyphenyl)glyoxal: reaction rates and intermediates

Reaction rates and pathways of two commonly used arginine-modifying reagents, phenylglyoxal (PGO) and (p-hydroxyphenyl)glyoxal (HPGO), were investigated by spectrophotometry. The initial rate at pH 9.0 of PGO with arginyl compounds was found to be 15 to 20 times greater than that of HPGO in the absence of borate but only 1.6 times greater in the presence of borate. Time-resolved spectra of HPGO reactions with arginines revealed, in contrast to the relatively simple spectra of PGO, at least two spectrophotometrically identifiable intermediates, one non-absorbing at 336 nm and the other absorbing at 458 nm. The 458 nm absorbing intermediate species was no longer detectable in the presence of borate.     

Stimulating action of polyphosphates on peptide formation from glycine and phenylalanine amides under abiogenic conditions

Synthesis of peptides during polymerization of GlyNH2 and PheNH2 has been demonstrated by means of gel-chromatography and thin-layer chromatography. The optima of pH and temperature have been estimated for the reaction. Grem's salt, tripolyphosphate and pyrophosphate were shown to cause the stimulatory effect on the peptide formation, but there was no correlation between the yield of the peptides and the hydrolysis of the polyphosphates. On the basis of the experimental data, it has been concluded that hydrolysis of polyphosphates is not an energy source for the polymerization of GlyNH2 and PheNH2. Therefore, polyphosphates cause catalytic effect on the peptide synthesis from the amides of amino acids in homogeneous medium.     

On the lack of formation of l-(+)-3-hydroxybutyrate by liver

The terminal carbon of palmitic acid, traced with ¹⁴C, is preferentially incorporated into carbon 4 of hydroxybutyrate formed by hepatocytes and perfused livers from 18- to 19-day-old rats and perfused livers from fasted adult rats. However, ¹⁴C from [13-¹⁴C]palmitic acid is incorporated into carbon 1 of the hydroxybutyrate to the same extent as any one of the first 12 carbons of palmitic acid as assessed with [1-¹⁴C]palmitic acid and [6-¹⁴C]palmitic acid. Therefore, the hydroxybutyrate is formed via hydroxymethylglutaryl-CoA, i.e., it is in the d configuration, and hydrolysis of l-hydroxybutyryl-CoA, the intermediate in the β oxidation of the palmitate, does not occur. Further, a negligible amount of ¹⁴C remains in hydroxybutyrate formed from ¹⁴C-labeled palmitic acid by isolated hepatocytes and perfused livers from the young rats, when the hydroxybutyrate is treated with d-(?)-3-hydroxybutyrate dehydrogenase to convert the d isomer to acetoacetate. Thus, l-(+)-3-hydroxybutyrate is not produced by rat liver as assessed using these preparations.     

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.     

Mechanism of the glycine cleavage reaction. Properties of the reverse reaction catalyzed by T-protein

T-protein, one of the components of the glycine cleavage system, catalyzes the synthesis of the H-protein-bound intermediate from methylenetetrahydrofolate, ammonia, and H-protein having a reduced lipoyl prosthetic group (Okamura-Ikeda, K., Fujiwara, K., and Motokawa, Y. (1982) J. Biol. Chem. 257, 135-139). Spectroscopic studies indicated that the utilization of methylenetetrahydrofolate occurred only in the presence of the three substrates, indicating the formation of a quaternary complex. The amount of methylenetetrahydrofolate consumed was equal to that of methylene carbon attached to H-protein. Steady-state kinetic studies show that the reaction proceeds through an Ordered Ter Bi mechanism. Reduced H-protein is the first substrate that binds T-protein followed by methylenetetrahydrofolate and ammonia. The order of release of products is tetrahydrofolate and the H-protein-bound intermediate. Km values for H-protein, methylenetetrahydrofolate, and ammonia are 0.55 microM, 0.32 mM, and 22 mM, respectively.     

Rapid and reliable peptide de novo sequencing facilitated by microfluidic chip-based Edman degradation

This paper expands the application of the newly developed highly sensitive microfluidic chip-based Edman degradation system. Comparison between the MS/MS spectra of a native peptide and its N-terminus truncated counterpart after carrying out one cycle of Edman degradation in a microfluidic chip can not only provide N-terminal residue information, but also facilitate the identification of different series of fragment ions. Manual peptide sequencing is more feasible and rapid using this method as demonstrated with three peptide examples including one neuropeptide. Furthermore, two cycles of Edman degradation allow the determination of the exact value of b 2 ion of the intact peptide, which can serve as an internal calibrant to increase the mass accuracy of the MS/MS spectrum.     

A novel control of enzymatic enantioselectivity through the racemic temperature influenced by reaction media

The influence of reaction media on the racemic temperature (T_r) in the lipase-catalyzed resolution of ketoprofen vinyl ester was investigated. An effective approach to the control of the enzymatic enantioselectivity and the prediction of the increasing tendency was developed based on the T_r influenced by reaction media. The T_r for the resolution catalyzed by Candida rugosa lipase (CRL) was found at 29 °C in aqueous and S-ketoprofen was obtained predominantly at 40 °C. However, CRL showed R-selectivity at 40 °C in diisopropyl ether because the T_r was changed to 56 °C. CRL, lipase from AYS Amano^® and Mucor javanicus lipase were further applied for the investigation of the enzymatic enantioselectivity in dioxane, DIPE, isooctane and their mixed media with water. The effects of the reaction medium on T_r could be related to the solvent hydrophobicity, the lipase conformational flexibility and the interaction between the enantiomers and the lipase.     

Protein misfolding and amyloid formation for the peptide GNNQQNY from yeast prion protein Sup35: simulation by reaction path annealing

We study the early steps of amyloid formation of the seven residue peptide GNNQQNY from yeast prion-like protein Sup35 by simulating the random coil to beta-sheet and alpha-helix to beta-sheet transition both in the absence and presence of a cross-beta amyloid nucleus. The simulation method at atomic resolution employs a new implementation of a Langevin dynamics "reaction path annealing" algorithm. The results indicate that the presence of amyloid-like cross-beta-sheet strands both facilitates the transition into the cross-beta conformation and substantially lowers the free energy barrier for this transition. This model systems allows us to investigate the energetic and kinetic details of this transition, which is consistent with an auto-catalyzed, nucleation-like mechanism for the formation of beta-amyloid. In particular, we find that electrostatic interactions of peptide backbone dipoles contribute significantly to the stability of the beta-amyloid state. Furthermore, we find water exclusion and interactions of polar side-chains to be driving forces of amyloid formation: the cross-beta conformation is stabilized by burial of polar side-chains and inter-residue hydrogen bonds in the presence of an amyloid-like "seed". These findings are in support of a "dry, polar zipper model" of amyloid formation.