Intramolecular general acid catalysis in the binding reactions of α2 and complement components C3 and C4

The complement system proteins C3 and C4 and the plasma protease inhibitor alpha 2-macroglobulin, when activated by limited proteolysis, can bind covalently to other macromolecules. The three proteins also exhibit an unusual internal peptide-bond cleavage reaction when denatured. The covalent binding reaction is likely to occur by a transacylation mechanism involving an internal thiolester in the three proteins. However, the activated species of these proteins are much more reactive than simple thiolesters. Studies of molecular models of the thiolester region in C3 show that an intramolecular acid catalysis mechanism can both account for the exceptional reactivity of the activated form of these proteins and provide an explanation for the denaturation-induced peptide bond cleavage.     

The role of hydrogen bonding in the enzymatic reaction catalyzed by HIV-1 protease

The hydrogen-bond network in various stages of the enzymatic reaction catalyzed by HIV-1 protease was studied through quantum-classical molecular dynamics simulations. The approximate valence bond method was applied to the active site atoms participating directly in the rearrangement of chemical bonds. The rest of the protein with explicit solvent was treated with a classical molecular mechanics model. Two possible mechanisms were studied, general-acid/general-base (GA/GB) with Asp 25 protonated at the inner oxygen, and a direct nucleophilic attack by Asp 25. Strong hydrogen bonds leading to spontaneous proton transfers were observed in both reaction paths. A single-well hydrogen bond was formed between the peptide nitrogen and outer oxygen of Asp 125. The proton was diffusely distributed with an average central position and transferred back and forth on a picosecond scale. In both mechanisms, this interaction helped change the peptide-bond hybridization, increased the partial charge on peptidyl carbon, and in the GA/GB mechanism, helped deprotonate the water molecule. The inner oxygens of the aspartic dyad formed a low-barrier, but asymmetric hydrogen bond; the proton was not positioned midway and made a slightly elongated covalent bond, transferring from one to the other aspartate. In the GA/GB mechanism both aspartates may help deprotonate the water molecule. We observed the breakage of the peptide bond and found that the protonation of the peptidyl amine group was essential for the peptide-bond cleavage. In studies of the direct nucleophilic mechanism, the peptide carbon of the substrate and oxygen of Asp 25 approached as close as 2.3 A.     

The Synthesis of Phosphopeptides Using Microwave-assisted Solid Phase Peptide Synthesis

A series of phosphorylated peptides were synthesised using microwave mediated solid phase peptide synthesis. Acidic cleavage of peptides from the solid support using microwave irradiation often resulted in reattachment of the phosphate benzyl protecting group to the peptide chain. However for most phosphopeptide sequences performing the cleavage reaction at room temperature in order to minimize this undesired alkylation was successful. Notably for phosphopeptides containing a methionine residue flanking the phosphorylated residue (for serine and threonine) the trifluoroacetic acid mediated cleavage afforded the benzylated side product as a major component. This detrimental process was not observed for a corresponding tyrosine containing sequence.     

Rabbit tissue peptidases that hydrolyse the peptide hormone bradykinin. Differences in enzymic properties and concentration in rabbit tissues.

The distribution and properties of neutral peptidases acting on the peptide hormone bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) were determined in several rabbit tissues. The supernatant and particulate fractions prepared from tissue homogenates (25000g for 60min) were studied. Bradykinin inactivation (kininase activity) was measured by bioassay with the isolated guinea-pig ileum. The sites of peptide-bond cleavage were determined in the amino acid analyser, which permits detection and measurement of amino acids and peptides derived from bradykinin. The results indicate that kininases are present in a wide range of concentrations in different tissues, kidney and lung having the most activity. Kininases present in different tissues were distinguished on the basis of sensitivity to the effects of EDTA, dithiothreitol and ZnCl2 and by the site of peptide-bond hydrolysis in bradykinin.     

Hydrolysis of DNA by a Branched Peptide without Aromatic Residues

A branched peptide Nα, Nɛ-di (l-leucyl)-l-lysine was found to efficiently cleave supercoiled double-strand DNA such as PUC19 DNA at optimum pH 4.0 in 40 mmol/l Britton–Robbinson buffer. The T4 ligase experiment implied that the DNA cleavage occurs via a hydrolytic path. The dependence of the cleavage reaction on the ionic strength indicated that the interaction of DNA with the branched peptide involve only electrostatic binding.     

Action of human liver cathepsin B on the oxidized insulin B chain.

The lysosomal cysteine proteinase cathepsin B (from human liver) was tested for its peptide-bond specificity against the oxidized B-chain of insulin. Sixteen peptide degradation products were separated by high-pressure liquid chromatography and thin-layer chromatography and were analysed for their amino acid content and N-terminal amino acid residue. Five major and six minor cleavage sites were identified; the major cleavage sites were Gln(4)-His(5), Ser(9)-His(10), Glu(13)-Ala(14), Tyr(16)-Leu(17) and Gly(23)-Phe(24). The findings indicate that human cathepsin B has a broad specificity, with no clearly defined requirement for any particular amino acid residues in the vicinity of the cleavage sites. The enzyme did not display peptidyldipeptidase activity with this substrate, and showed a specificity different from those reported for two other cysteine proteinases, papain and rat cathepsin L.     

The effect of proteinases on phenylalanine ammonia-lyase from the yeast Rhodotorula glutinis.

Phenylalanine ammonia-lyase (EC 4.3.1.5) of the yeast Rhodotorula glutinis was rapidly inactivated by duodenal juice. It was susceptible to chymotrypsin and subtilisin and to a lesser extent trypsin. Initial proteolysis of the enzyme by chymotrypsin and trypsin resulted in cleavage of the monomeric subunit (75 000 Mr) into a large (65 000 Mr) and a small (10 000 Mr) peptide. The small peptide was rapidly degraded. The 65 000-Mr fragment was resistant to prolonged incubation with chymotrypsin, but was degraded by trypsin under the same conditions. Phenylalanine ammonia-lyase was cleaved into several polypeptides by subtilisin, the 65 000-Mr peptide being totally absent. The N-terminal region of the enzyme was contained in the 65 000-Mr fragment, as was the dehydroalanine moiety, the prosthetic group. Active-site-binding ligands protect the enzyme from inactivation by the three proteinases, and peptide-bond cleavage by trypsin and chymotrypsin. Several chemical modifications were performed on phenylalanine ammonia-lyase. Some decreased its antigenicity, and ethyl acetimidate decreased the rate of degradation of the 65 000-Mr peptide by trypsin. The modification did not protect the enzyme from proteolytic inactivation of the enzymic activity. These observations are discussed in terms of the structure of phenylalanine ammonia-lyase and site of action of the proteinases.     

Autolytic fragmentation of complement components C3 and C4 under denaturing conditions, a property shared with alpha 2-macroglobulin.

The alpha polypeptide chain of the complement protein C3 splits into two fragments of 74 000 and 46 000 apparent mol.wt. under certain conditions used to prepare the protein for SDS (sodium dodecyl sulphate)/polyacrylamide-gel electrophoresis. The cleavage reaction occurs over a wide range of temperatures and from pH 4.6 to 10.6 in the presence of denaturants such as urea, SDS and guanidine hydrochloride. It is also induced by heat-denaturation of C3 in the absence of chemical denaturants. The reaction occurs only with haemolytically active C3, and is not observed with hydroxylamine-inactivated C3 or with C3b. A similar cleavage of the alpha-chain of complement component C4 occurs under the same conditions, forming fragments of 53 000 and 41 000 apparent mol.wt. This reaction is again specific for haemolytically active C4, and does not occur with C4b or hydroxylamine-inactivated C4. The complement component C5, although structurally similar to C3 and C4, does not undergo a reaction of this type. The characteristics of the denaturation-induced cleavage of C3 and C4 match those described for the 'heat-induced' cleavage of alpha 2-macroglobulin [Harpel, Hayes & Hugli (1979) J. Biol. Chem. 254, 8669-8678]. Cleavage of alpha 2-macroglobulin is also specific for the active form of the protein, and does not occur with chemically inactivated or proteinase-cleaved forms. The unusual conditions and specificity of the peptide-bond cleavage in all three proteins suggest that it is an autolytic process rather than being the result of trace proteinase contamination. The active forms of C3, C4 and alpha 2-macroglobulin have the transient ability to form covalent bonds after activation. The autolytic cleavage reaction is likely to be related to the covalent-bond-forming reactions of these proteins.     

How ribosomes make peptide bonds

Ribosomes are molecular machines that synthesize proteins in the cell. Recent biochemical analyses and high-resolution crystal structures of the bacterial ribosome have shown that the active site for the formation of peptide bonds--the peptidyl-transferase center--is composed solely of rRNA. Thus, the ribosome is the largest known RNA catalyst and the only natural ribozyme that has a synthetic activity. The ribosome employs entropic catalysis to accelerate peptide-bond formation by positioning substrates, reorganizing water in the active site and providing an electrostatic network that stabilizes reaction intermediates. Proton transfer during the reaction seems to be promoted by a concerted shuttle mechanism that involves ribose hydroxyl groups on the tRNA substrate.     

Reconstruction of peptidyltransferase activity on 50S and 70S ribosomal particles by peptide fragments of protein L16

Ribosomal protein L16 was digested with Staphylococcus aureus protease V8 and the resulting peptides were separated by reversed-phase high-performance liquid chromatography. One of the fragments, identified by sequence analysis as the N-terminal peptide of L16, was shown to exhibit partial peptide-bond-formation and transesterification activities of peptidyltransferase upon reconstitution with L16-depleted 50S core particles. However, several proteins enhanced these activities. L15 increased both reactions when added to the reconstitution mixture, suggesting a limited capacity of the L16 peptide to incorporate into 50S core particles. In contrast, the interaction of L11 with the N-terminal peptide stimulated the transesterification reaction but not the peptide-bond-forming activity of ribosomes, indicating a different topological domain for these reactions. Also, EF-P, a soluble protein which reconstructs the peptide-bond formation and transesterification reactions on 70S ribosomes, stimulated both peptidyltransferase activities exhibited by the L16 N-terminal peptide.     

Tryptic Hydrolysis of Dodecapeptides Possessing Paired Basic Residues

Four dodecapeptides of general formula Tyr-Gly-Gly-Phe-Met-X-X-Tyr-Gly-Gly-Phe-Met-NH₂ (Enk-X-X-Enk-NH₂) possessing X = Arg or Lys have been synthesized and subjected to cleavage by trypsin. The peptide with the sequence containing -Lys-Arg-, depicted as BI-NH₂, represents the 100–111 segment of proenkephalin. The time course of the degradation was followed by high performance liquid chromatography. This method allows one to observe the formation of not only the final but also intermediate peptides. Among the peptides studied, the most susceptible to the cleavage was BI-NH₂. The primary hydrolysis proceeded rapidly at the arginine residue, followed by slow release of arginine. The other peptides (with -Arg-Arg-, -Lys-Lys- and -Arg-Lys-) were cleaved at both possible positions, but the resulting mixture contained Enk-X as a major product, which was the result of both primary and secondary cleavage.     

Tryptic Hydrolysis of Dodecapeptides Possessing Paired Basic Residues

Summary Four dodecapeptides of general formula Tyr-Gly-Gly-Phe-Met-X-X-Tyr-Gly-Gly-Phe-Met-NH₂ (Enk-X-X-Enk-NH₂) possessing X-Arg or Lys have been synthesized and subjected to cleavage by trypsin. The peptide with the sequence containing-Lys-Arg-, depicted as BI-NH₂, represents the 100–111 segment of proenkephalin. The time course of the degradation was followed by high performance liquid chromatography. This method allows one to observe the formation of not only the final but also intermediate peptides. Among the peptides studied, the most susceptible to the cleavage was BI-NH₂. The primary hydrolysis proceeded rapidly at the arginine residue, followed by slow release of arginine. The other peptides (with-Arg-Arg-,-Lys-Lys-and-Arg-Lys-) were cleaved at both possible positions, but the resulting mixture contained Enk-X as a major product, which was the result of both primary and secondary cleavage.     

Identification of alternative products and optimization of 2-nitro-5-thiocyanatobenzoic acid cyanylation and cleavage at cysteine residues

The reagent 2-nitro-5-thiocyanatobenzoic acid (NTCB) is commonly used to cyanylate and cleave proteins at cysteine residues, but this two-step reaction requires lengthy incubations and produces highly incomplete cleavages. In previous reports, incomplete cleavage was attributed to a competing beta-elimination reaction that converts cyanylated cysteine to dehydroalanine. In this study, previously unidentified side reactions of the NTCB cleavage were discovered and beta-elimination was not the major reaction competing with peptide bond cleavage. A major side reaction was identified as carbamylation of lysine residues. Carbamylation could be minimized by desalting the cyanylation reaction before cleavage or by reducing the reactant concentrations, but both methods suffered from further reductions in cleavage efficiency. Based on model peptide studies, poor cleavage was primarily caused by a mass neutral rearrangement of the cyanylated cysteine which produced a cleavage-resistant, nonreducible product. The formation of this product could be minimized by using stronger nucleophiles for the cleavage reaction. We discovered that base-catalyzed nucleophilic cleavage could be achieved with many amino-containing compounds. Most notably, glycine is capable of promoting efficient cleavage. In addition, efficient NTCB cleavage can be performed in a simple one-step method without a prior cyanylation step, rather than the previously described two-step reaction.     

Microwave-mediated enzymatic modifications of DNA

Here we report microwave-induced specific cleavage, ligation, dephosphorylation, and phosphorylation of nucleic acids catalyzed by restriction endonucleases, T4 DNA ligase, T4 polynucleotide kinase, and calf intestinal alkaline phosphatase. The microwave-mediated method has dramatically reduced the reaction time to 20 to 50 s. In control experiments, the same reactions failed to give the desired reaction products when carried out in the same time periods but without microwave irradiation. Because the microwave method is rapid, it could be a useful alternative to the time-consuming conventional procedure for enzymatic modification of DNA.     

Microwave‐assisted TFA cleavage of peptides from Merrifield resin

Microwave‐assisted (MW) reactions are of special interest to the chemical community due to faster reaction times, cleaner reactions and higher product yields. The adaptation of MW to solid phase peptide synthesis resulted in spectacular syntheses of difficult peptides. In the case of Merrifield support, used frequently in synthesis of special peptides, the conditions used in product cleavage are not compatible with off‐resin monitoring of the reaction progress. The application of MW irradiation in product removal from Merrifield resin using trifluoroacetic acid (TFA) was investigated using model tetrapeptides and the effects were compared with standard trifluoromethanesulphonic acid (TFMSA) cleavage using elemental analysis as well as chromatographic (HPLC) and spectroscopic (IR) methods. The deprotection of benzyloxycarbonyl and benzyl groups in synthetic bioactive peptides was analyzed using LC‐MS and MS/MS experiments. In a 5 min microwave‐assisted TFA reaction at low temperature, the majority of product is released from the resin, making the analytical scale MW‐assisted procedure a method of choice in monitoring the reactions carried out on Merrifield resin due to the short reaction time and compatibility with HPLC and ESI‐MS conditions. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Kinetics of chymotrypsin- and papain-catalysed synthesis of [leucine]enkephalin and [methionine]enkephalin.

The proteinase-catalysed synthesis of [Leu]enkephalin and [Met]enkephalin was studied kinetically. N alpha-t-Butoxycarbonyl-amino acids and peptides or their ethyl esters served as acyl donors, and amino acid phenylhydrazides were used as acyl acceptors. Initial-velocity measurements of alpha-chymotrypsin-catalysed peptide synthesis gave rise to kinetic patterns that are compatible with a ping-pong mechanism modified by a hydrolytic branch. Initial-rate and alternative-substrate inhibition patterns for papain-controlled peptide-bond formation are consistent with a sequential ordered mechanism with the acyl donor as the obligatory first substrate. On the basis of the observed kinetic features, reaction mechanisms are proposed for chymotrypsin- and papain-catalysed peptide synthesis that inversely equal those describing the pathways of proteolysis. The respective initial-velocity expressions for bireactant systems are given, along with the numerical values of the corresponding kinetic parameters.     

Chymotrypsin-catalyzed peptide synthesis. Kinetic analysis of the kinetically controlled peptide-bond formation

The kinetics of peptide-bond formation catalyzed by delta-chymotrypsin has been studied for a number of peptide products of different length using fixed concentrations of the acyl component (Ac-Phe-OMe, Ac-Ala-Ala-Phe-OMe, or Ac-Ala-Ala-Tyr-OMe) and varying concentration of the amino component (H-Ala-NH2 or H-Ala-Ala-NH2). The time course of the reactions was followed by monitoring ester consumption and peptide product formation by analytical HPLC. On the basis of a plausible four-centre mechanistic model, the theoretical time course of these reactions was calculated using rate and equilibrium constants determined by separate kinetic experiments. The excellent agreement observed between the theoretical and the experimental time courses supports the proposed mechanism and provides evidence for the validity of the present kinetic approach. By focusing attention on the rate constants which are critical for efficient synthesis, this mechanistic information constitutes a valuable basis for the use of the enzymatic peptide synthesis in preparative applications.     

C-terminal N-alkylated peptide amides resulting from the linker decomposition of the Rink amide resin: a new cleavage mixture prevents their formation.

Decomposition of the resin linkers during TFA cleavage of the peptides in the Fmoc strategy leads to alkylation of sensitive amino acids. The C-terminal amide alkylation, reported for the first time, is shown to be a major problem in peptide amides synthesized on the Rink amide resin. This side reaction occurs as a result of the Rink amide linker decomposition under TFA treatment of the peptide resin. The use of 1,3-dimethoxybenzene in a cleavage cocktail prevents almost quantitatively formation of C-terminal N-alkylated peptide amides. Oxidized by-product in the tested Cys- and Met-containing peptides were not observed, even if thiols were not used in the cleavage mixture.     

Salt-assisted acid hydrolysis of chitosan to oligomers under microwave irradiation

The effect of inorganic salts such as sodium chloride on the hydrolysis of chitosan in a microwave field was investigated. While it is known that microwave heating is a convenient way to obtain a wide range of products of different molecular weights only by changing the reaction time and/or the radiation power, the addition of some inorganic salts was shown to effectively accelerate the degradation of chitosan under microwave irradiation. The molecular weight of the degraded chitosan obtained by microwave irradiation was considerably lower than that obtained by traditional heating. Moreover, the molecular weight of degraded chitosan obtained by microwave irradiation assisted under the conditions of added salt was considerably lower than that obtained by microwave irradiation without added salt. Furthermore, the effect of ionic strength of the added salts was not linked with the change of molecular weight. FTIR spectral analyses demonstrated that a significantly shorter time was required to obtain a satisfactory molecular weight by the microwave irradiation-assisted inorganic salt method than by microwave irradiation without inorganic salts and conventional technology.     

Requirements for the initiation of polyphenylalanine synthesis by recombined ribosomal subunits from yeast

A reaction stimulated by elongation factor 2 (EF-2) is necessary for the formation of the initial peptide-bond in poly(U)-directed peptide synthesis on yeast ribosomal subunits. The stimulation is inhibited by fusidic acid or diphtheria toxin together with NAD. It is assumed that the reaction of EF-2 represents translocation: in the presence of EF-2 almost twice the amount of N-acetyl-phenylalanyl-tRNA is bound to ribosomes and moreover, upon addition of puromycin a considerable stimulation of N-acetyl-phenylalanyl-puromycin formation is found. Contradictory data in the literature on this subject may be due to nonenzymatic translocation caused by a high monovalent cation concentration in the ribosome preparations or to an unspecific method for the extraction of N-acetyl-phenylalanyl-puromycin.