Peptide synthesis catalyzed by proteases. Elevated nucleophilic activity of naphthylamides of amino acids in acyl transfer reactions catalyzed by alpha-chymotrypsin

It was found that the reactivity of alpha-amino acid naphthylamides in acyl transfer reactions catalyzed by alpha-chymotrypsin exceeds by more than two orders of magnitude the effective reactivity of other C-protected derivatives of these compounds. A detailed kinetic analysis of the acyl transfer of the tert-butyl oxycarbonyl-L-methionine residue from its p-nitrophenyl ester to L-arginine naphthylamide was carried out. A minimal kinetic scheme of acyl transfer reactions is proposed, including together with the major process, i.e., acyl residue transfer to the nucleophil, the hydrolysis of the acyl enzyme-nucleophil complex and nucleophil binding by the free enzyme. The numeric values of some kinetic constants were determined. A theoretical analysis of the effect of hydrolysis of the acyl enzyme-nucleophil complex on the degree of nucleophil conversion into the peptide at initial acyl group donor and nucleophil concentrations was carried out.     

Peptide bond synthesis by clostridiopeptidase B

Summary Clostridiopeptidase B was used for peptide bond synthesis with Cbz-ARG-OMe as substrate and 17 amino acid amides as nucleophiles. Synthetic yields ranging from 75 to 98% were obtained for most of the amides.N.R.C.C. #26185.     

Peptide synthesis catalyzed by papain at alkaline pH values

The synthesis of peptides in the presence of papain at pH 8-9.5 is described. Starting substances are acylamino acid alkyl esters (the carboxyl component) and amides or tert.-butylesters of amino acids, as well as peptide (the amino component). Under such conditions secondary hydrolysis is not essential, making the synthesis of peptides soluble in aqueous medium. The yield of peptides is 50-94%. The effect of different factors (temperature, solvents, reagent concentrations) on the result of the reaction has been studied. It has been found that an excess of the carboxyl component is expedient to increase the yield of peptides.     

Peptide synthesis catalyzed by subtilisin-72 in organic solvents

The solubility, stability, and activity of native subtilisin 72 and of its complex with SDS were comparatively studied in a number of polar organic solvents. Subtilisin was found to catalyze peptide bond formation when suspended in acetonitrile or solubilized as a complex with SDS in ethanol and isopropanol. Tripeptide Z-Ala-Ala-Leu-pNA, tetrapeptides A-Ala-Ala-P1-P1'-B, where A = Z or Abz; P1 = Leu, Phe, Met, Trp, Ile, Tyr, Phe(NO2), or Glu(OMe), P1' = Leu, Phe, Glu, Ala, Ile, Val, or Arg; B = NH2, pNA, or 2-(2,4-dinitrophenyl)aminoethylamine residue (Ded); pentapeptides Z-Ala-Ala-Leu-Ala-Ala-pNA and Z-Ala-Ala-Leu-Ala-Phe-pNA; and hexapeptide Abz-Val-Ala-Phe-Phe-Ala-Ala-Ded were synthesized using the SDS-subtilisin complex. The complex also efficiently catalyzed the oligomerization of tripeptide H-Phe-Ala-Leu-OCH3 in ethanol, which resulted in a 63:37 mixture of trioligomer and tetraoligomer. It was demonstrated that SDS-subtilisin is a much more efficient catalyst than the suspension of native enzyme.     

Peptide synthesis catalyzed by proteases. The effect of temperature on kinetics of acyl transfer catalyzed by papain

The effect of temperature on the kinetics of papain-catalyzed peptide synthesis was studied. A characteristic feature of this process is that the peptide synthesis is accompanied both by the hydrolysis of the synthesized bond and by the further elongation of the peptide chain. These phenomena yield the maximum on the time dependence of the first synthetic product. A decrease in temperature is an effective way to increase the yield of this substance which reflects the increase in the nucleophil reactivity resulting from the temperature decrease. Moreover, the latter diminishes the contribution of secondary enzymatic reactions such as further hydrolysis of the reaction product and polypeptide chain elongation.     

Peptide bond formation mediated by substrate mimetics. Structure-guidedoptimization of trypsin for synthesis.

Substrate mimetics are excellent tools for protease-mediated peptide synthesis that enable the coupling of peptides independently of the primary specificity of the enzyme without undesired cleavages of the newly formed peptide bonds. However, the synthetic utility of this beneficial approach is limited to reactions with nonspecific amino-acid-containing peptides while the coupling of specific ones leads to unwanted cleavages due to the native proteolytic activity of the biocatalyst. This paper reports on the use of site-directed mutagenesis to design trypsin variants with decreased cleavage activity. Starting from the variant D189S, which is known for its low proteolytic potential, Ser189 and Ser190 were exchanged for Ala to further repress the inherent amidase activity of trypsin D189S. The effect of mutations was analysed by model synthesis reactions using specific amino-acid-containing peptides and substrate mimetics as the reactants. Finally, computer-assisted protein-ligand docking studies were performed to get closer insight into the molecular basis of the experimental results.     

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.     

Peptide synthesis catalyzed by Cynara cardunculus L. Acid protease in aqueous-organic biphasic systems

The ability of Cynara cardunculus L. protease to perform peptide synthesis was investigated using an aqueous organic biphasic system and the amino acid derivatives CBZ.Phe and Met.OMe. The reaction products were separated by RP-HPLC and identified by amino acid analysis and by EI-MS. Significant amounts of the dipeptide CBZ.Phe.Met.OMe was produced within 24h by Cynara cardunculus L. protease as compared with the amount produced by pepsin under the same conditions.     

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.     

Kinetics of amino acid esterification catalyzed by hydrophobic derivatives of alpha-chymotrypsin

Hexyl-alpha-chymotrypsin, a hydrophobic derivative of the enzyme, is explored for the proteinase-catalyzed ester synthesis reaction with N-acetyl-L-tyrosine and ethanol. To guarantee the preservation of the enzyme activity and to allow for the extraction of the product in the organic phase, a biphasic system was used. The Vm increased for the modified enzyme. This phenomenon was linked to the modification of the protein as demonstrated by its chemical denaturation with sodium dodecyl sulfate.     

Peptide bond synthesis: function of the efp gene product

The efp gene encodes a protein that is essential for the growth and for the viability of Escherichia coli cells. Interruption of this gene results in cell death due to a defect in protein synthesis. We report here that the EFP protein, encoded by the efp gene, is required for in vitro reconstitution of polypeptide synthesis in a system programmed by a native template which contains each of the purified initiation factors, IF1, IF2, IF3; the elongation factors, EFTu, EFTs and EFG, and a protein called W that is required to eject tRNAs from ribosomes. The EFP protein is required for enhancing the rate and the extent of synthesis in the presence of all of the above factors. The EFP protein stimulates synthesis of poly(Phe) programmed with poly(rU) only if N-acetyl Phe-tRNA initiates the reactions under conditions that foster the dissociation of the 70S ribosome. Study of the ability of the ribosome to synthesize a number of fMet-initiated dipeptides from CCA amino acyl acceptors suggests that EFP acts to promote synthesis with acceptors that are poor donors for the the reconstituted peptidyl transferase.     

Peptide and ester synthesis in organic solvents catalyzed by seryl proteases linked to alumina

Trypsin and alpha-chymotrypsin were immobilized to alumina-phosphocolamine complex, activated by glutaraldehyde. The immobilized enzymes show a great stability toward organic solvents miscible or immiscible with water. In the presence of a low concentration of water, the immobilized enzymes catalyzed transesterification reactions as well as peptide synthesis. The synthesized peptides were stable toward the immobilized enzymes.     

Peptide synthesis catalyzed by proteases. Analysis of a kinetic model for enzymes with acyl-enzyme mechanism of action

The kinetics of peptide synthesis via transfer of the acyl moiety from activated derivatives of amino acids or peptides (S) to nucleophiles (N) catalyzed by proteases forming an acyl-enzyme intermediate, was analysed. A kinetic model assumes enzymatic hydrolysis of the formed peptide (P), so the kinetic curve for P has a maximum (denoted as pmax). Particular attention was given to the analysis of the effects of the initial concentrations and kinetic constants on pmax. Computer analysis demonstrated that at a given ratio of initial S and N concentrations pmax is affected only by the ratio of the second order rate constants for enzymatic hydrolysis of S and P (alpha) and the ratio of rate constants for an attack of the acyl-enzyme intermediate by nucleophile and water (beta). These conclusions apply regardless of the existence of enzyme forms other than a free enzyme and an acyl-enzyme intermediate. Thus, the kinetically controlled maximum yield of peptide (pmax) can be calculated a priori from the values of alpha and beta which can be readily evaluated from the reference data. Simple explicit expressions were obtained, allowing fairly accurate prediction of pmax for a broad spectrum of S and N initial concentrations.     

Peptide bond formation stimulated by protein synthesis factor EF-P depends on the aminoacyl moiety of the acceptor.

Elongation factor EF-P is a soluble protein that stimulates peptide bond synthesis catalyzed by the 50-S ribosomal subunit. This factor was previously identified and characterized based on its ability to promote the synthesis of formylmethionine-puromycin. In the present work, we tested the ability of EF-P to promote peptide bond synthesis between ribosome-bound fMet-tRNA and several analogues of the 3' terminus of aminoacyl-tRNA, i.e. the cytidylyl(3'-5')-[2'(3')-O-L-aminoacyladenosines]. EF-P promoted synthesis to the greatest extent with certain acceptors which were otherwise inefficient in the peptidyl transferase reaction. This activity of EF-P could not be replaced by the other soluble proteins known to be involved in polypeptide synthesis, such as EF-Tu, EF-Ts and EF-G. One role of EF-P in protein synthesis may be to allow peptide bond synthesis to occur more efficiently with some aminoacyl-tRNAs that are poor acceptors for the ribosomal peptidyl transferase.     

Solid-phase peptide synthesis by ion-paired alpha-chymotrypsin in nonaqueous media

Solid-phase synthesis of dipeptides in low-water media was achieved using AOT ion-paired alpha-chymotrypsin solubilized in organic solvents. Multiple solvents and systematic variation of water activity, a(w), were used to examine the rate of coupling between N-alpha-benzyloxycarbonyl-L-phenylalanine methyl ester (Z-Phe-OMe) and leucine as a function of the reaction medium for both solid-phase and solution-phase reactions. In solution, the observed maximum reaction rate in a given solvent generally correlated with measures of hydrophobicity such as the log of the 1-octanol/water partitioning coefficient (log P) and the Hildebrand solubility parameter. The maximum rate for solution-phase synthesis (13 mmol/h g-enzyme) was obtained in a 90/10 (v/v) isooctane/tetrahydrofuran solvent mixture at an a(w) of 0.30. For the synthesis of dipeptides from solid-phase leucine residues, the highest synthetic rates (0.14-1.3 mmol/h g-enzyme) were confined to solvent environments that fell inside abruptly defined regions of solvent parameter space (e.g., log P > 2.3 and normalized electron acceptance index <0.13). The maximum rate for solid-phase synthesis was obtained in a 90/10 (v/v) isooctane/tetrahydrofuran solvent mixture at an a(w) of 0.14. In 90/10 and 70/30 (v/v) isooctane/tetrahydrofuran environments with a(w) set to 0.14, seven different N-protected dipeptides were synthesized on commercially available Tentagel support with yields of 74-98% in 24 h.     

Synthesis of N-protected peptide alcohols catalyzed by subtilisin or alpha-chymotrypsin in organic solvents

A series of N-protected peptide alcohols were synthesized using amino alcohols with unprotected hydroxy groups as amino components by the catalysis of subtilisin or alpha-chymotrypsin in organic solvents. N-protected aromatic amino acid esters were more suitable as acyl donors for subtilisin. The influences of different N-protecting groups, organic solvents, and content of water on synthesis of N-protected peptide alcohols were systematically studied.     

Dipeptide derivative synthesis catalyzed by Pseudomonas aeruginosa elastase.

Pseudomonas aeruginosa elastase was used to synthesize various N-protected dipeptide amides. The identity of the products was confirmed by FAB(+)-MS. After recrystallization, the yield of their synthesis was calculated, their purity was checked by RP-HPLC and their melting point was measured. With regard to the hydrolysis, it is well-established that the enzyme prefers hydrophobic amino acids in P'1 position and it has a wide specificity for the P1 position. This specificity was demonstrated to be quite unchanged when comparing the initial rates of peptide bond formation between different carboxyl donors (Z-aa) and nucleophiles (aa-NH2). The elastase, but not the thermolysin, was notably able to incorporate tyrosine and tryptophan in P'1 position. Furthermore, synthesis initial rates were at least 100 times faster with the elastase. To overcome the problematic condensation of some amino acids during chemical peptide synthesis, it has been previously suggested that enzymatic steps can combine with a chemical strategy. We demonstrated that the elastase readily synthesizes dipeptide derivatives containing various usual N-protecting groups. It was especially able to condense phenylalaninamide to Fmoc- and Boc-alanine. Increasing interest in peptides containing unnatural amino acids led us to try the elastase-catalyzed synthesis of Z-dipeptide amides including those amino acids in the P1 position. A synthesis was demonstrated with alphaAbu, Nle, Nva and Phg.     

3-Ribosyl-6-methyluracil as a phosphate acceptor in the synthesis of the internucleotide bond catalyzed by pancreatic ribonuclease.

The synthesis of uridylyl-(3'-5')-3-ribosyl-6-methyluracil (UprmU) catalyzed by pancreatic ribonuclease (EC 3.4.1.22) has been performed using uridine 2', 3'-cyclic phosphate (U greater than p) as phosphate donor and 3-ribosyl-6-methyluracil (rmU) as phosphate acceptor. The rate of synthesis of UprmU is much higher than that of uridylyl-(3'-5')-uridine (UpU) in a control experiment under the same conditions with uridine as acceptor. The yields of UpU and UprmU were 20 and 17% respectively. The competitive hydrolysis of the initial U greater than p also proceeds faster when rmU is used as the acceptor. The relationship between the conformation of this nucleoside and its acceptor activity in the enzymatic synthesis of the internucleotide bond is discussed.