Trypsin‐catalysed synthesis of oligopeptide amides: comparison of catalytic efficiency among trypsins of different origin (bovine, <i>streptomyces griseus</i> and chum salmon)

Sekizaki, Haruo; Itoh, Ken; Toyota, Eiji; Tanizawa, Kazutaka

doi:10.1002/psc.406

Cited by 6 publications

(3 citation statements)

References 30 publications

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“…In this way, 2-methyl Ala, 2-ethyl Ala and 2,2diethyl Ala could be used as acyl donors, in presence of bovine or bacterial trypsin. Similar results were obtained with other esters: para-(guanidinomethyl)phenyl- [41], guanidinonapthyl- [42], and with different proteases : trypsin of various origins (bovine, Streptomyces griseus, chum salmon [43,44]), thrombin [45]. Similar results were obtained with other esters: para-(guanidinomethyl)phenyl- [41], guanidinonapthyl- [42], and with different proteases : trypsin of various origins (bovine, Streptomyces griseus, chum salmon [43,44]), thrombin [45].…”

Section: Substrate Modification: Mimetic Sub-stratessupporting

confidence: 84%

Recent Trends in Protease-Catalyzed Peptide Synthesis

Lombard

Saulnier

Wallach

2005

PPL

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Enzymatic peptide syntheses may be either thermodynamically- or kinetically-controlled. The former may be catalyzed by any proteases; the latter is limited to serine and cysteine proteases. This methodology is quite stereospecific and avoids side chain protection but is suffering of some drawbacks. Thus, only two industrial processes are by now developed: the production of aspartame and the conversion of porcine into human insulin. However, recent improvements have been carried out in different directions: 1-Search for proteases with high and/or new P'1 and P1 specificities. 2-Protease engineering to promote synthesis towards hydrolysis and to enlarge specificity. 3-Development of mimetic or "inverse" substrates to limit further hydrolysis of synthesized peptide. 4-Change of the physical state of reactants. Three axes have mainly be explored: solid-solid conversion, use of cross-linked enzyme crystals (CLEC) and enzyme immobilization. 5-Modification of experimental conditions. The principal and recent developments deal with: heterogeneous catalysis, synthesis in low water-containing organic solvents, in ionic liquids or at subzero temperatures. This review will illustrate these new orientations with examples described in the recent literature.

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Section: Substrate Modification: Mimetic Sub-stratessupporting

confidence: 84%

Recent Trends in Protease-Catalyzed Peptide Synthesis

Lombard

Saulnier

Wallach

2005

PPL

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“…We have also studied trypsins from chum salmon (Oncorhynchus keta; Sekizaki et al, 2001Sekizaki et al, , 2002Toyota et al, 2002). The trypsins from chum salmon include seven anionic trypsins and one cationic trypsin.…”

Section: Introductionmentioning

confidence: 99%

A structural comparison of three isoforms of anionic trypsin from chum salmon (Oncorhynchus keta)

Toyota

Iyaguchi

Sekizaki

et al. 2009

Acta Crystallogr D Biol Cryst

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Three anionic salmon trypsin isoforms (CST-1, CST-2 and CST-3) were isolated from the pyloric caeca of chum salmon (Oncorhynchus keta). The order of catalytic efficiency (K(m)/k(cat)) of the isoforms during BAPA hydrolysis was CST-2 > CST-1 > CST-3. In order to find a structural rationalization for the observed difference in catalytic efficiency, the X-ray crystallographic structures of the three isoforms were compared in detail. Some structural differences were observed in the C-terminal alpha-helix, interdomain loop and active-site region. From the results of the detailed comparison, it appears that the structural flexibility of the C-terminal alpha-helix, which interacts with the N-terminal domain, and the substrate-binding pocket in CST-3 are lower than those in CST-1 and CST-2. In addition, the conformation of the catalytic triad (His57, Asp102 and Ser195) differs among the three isoforms. The imidazole N atom of His57 in CST-1 and CST-2 forms a hydrogen bond to the hydroxyl O atom of Ser195, but the distance between the imidazole N atom of His57 and the hydroxyl O atom of Ser195 in CST-3 is too great (3.8 A) for the formation of a hydrogen bond. Thus, the nucleophilicity of the hydroxyl group of Ser195 in CST-3 is weaker than that in CST-1 or CST-2. Furthermore, the electrostatic potential of the substrate-binding pocket in CST-2 is markedly lower than those in CST-1 and CST-3 owing to the negative charges of Asp150, Asp153 and Glu221B that arise from the long-range effect. These results may explain the higher catalytic efficiency of CST-2 compared with CST-1 and CST-3.

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“…13,14,[19][20][21][22][23][24][25][26][27][28] We also reported that a,a-dialkyl amino acid p-(guanidinomethyl)phenyl esters were useful substrates for enzymatic peptides synthesis containing such a hindered amino acid. 21) Recently, we reported that preparation of new two series inverse substrates such as N-Boc-amino acid p-and m-(amidinomethyl)phenyl esters.…”

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confidence: 96%

Trypsin-Catalyzed Synthesis of Dipeptide Containing .ALPHA.-Aminoisobutylic Acid Using p- and m-(Amidinomethyl)phenyl Esters as Acyl Donor

Sekizaki

Itoh

Shibuya

et al. 2008

CHEMICAL & PHARMACEUTICAL BULLETIN

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Sterically hindered a-amino acid such as the a-aminoisobutylic acid (Aib, 2-methylalanine) is one of the constituent of naturally occurring antibiotics.1-4) Peptides containing Aib are of interest as the model for conformational analysis of the peptide backbone (formation of a-or 3 10 -helices, or b-turns).5,6) However, the introduction of Aib into a peptide is difficult, 4,7,8) because the reactivity of sterically hindered a,a-disubstituted amino acid is much lower than that of typical a-amino acids. New chemical coupling reagent, 2-chloro-1,3-dimethylimidazolidium hexafluorophosphate, (CIP), for sterically hindered a,a-disubstituted amino acidis was developed and chemical synthesis of Alamethicin F-30 was achieved. 9)Peptide synthesis by protease-catalyzed reverse reaction has been extensively studied with a variety of amino acids and peptide derivatives as coupling. [10][11][12][13][14][15] The protease-catalyzed peptide synthesis is superior to the chemical coupling method. The method requires less-side chain protection than the chemical coupling method. A most serious drawback of the enzymatic method, however, is the respective substrate specificity. Thus, the application of proteases for peptide synthesis has not been fully exploited for possible synthesis of a number of biologically important peptides containing Damino acid or other unusual amino acid.In a previous paper, we reported that the p-amidinophenyl and p-guanidinophenyl esters behave as specific substrates for trypsin and trypsin-like enzymes such as thrombin and plasmin, etc. [16][17][18] In these esters the site-specific groups (charged amidinium and guanidinium) for the enzyme are included in the leaving-group portion instead of being in the acyl moiety. Such a substrate was termed "inverse substrate" by us.16) The esters provided a novel method for the specific introduction of an acyl group for a wide variety of acyl groups into a trypsin active site, and such acyl enzymes have proven to be useful for the peptide coupling. 13,14,[19][20][21][22][23][24][25][26][27][28] We also reported that a,a-dialkyl amino acid p-(guanidinomethyl)phenyl esters were useful substrates for enzymatic peptides synthesis containing such a hindered amino acid. 21)Recently, we reported that preparation of new two series inverse substrates such as N-Boc-amino acid p-and m-(amidinomethyl)phenyl esters. 29) In the present work, we investigated trypsin-catalyzed peptide synthesis using these synthetic substrates as acyl donor components. Comparison was made between two trypsins of different origin (bovine and Streptomyces griseus (SG) trypsin) as the catalyst for peptide synthesis as shown in Chart 1. Trypsin-Catalyzed Peptide Coupling ReactionThe trypsin-catalyzed peptide coupling reaction has been studied by using synhetic inverse substrates (1a-h, 2a-c) as acyl donors (Fig. 1) was examined in dimethyl sulfoxide (DMSO) or N,N-dimethylformamide (DMF) as a co-solvent. The reaction was also evaluated under the condition where the pH of the medium was changed and the ...

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Trypsin‐catalysed synthesis of oligopeptide amides: comparison of catalytic efficiency among trypsins of different origin (bovine, streptomyces griseus and chum salmon)

Cited by 6 publications

References 30 publications

Recent Trends in Protease-Catalyzed Peptide Synthesis

Recent Trends in Protease-Catalyzed Peptide Synthesis

A structural comparison of three isoforms of anionic trypsin from chum salmon (Oncorhynchus keta)

Trypsin-Catalyzed Synthesis of Dipeptide Containing .ALPHA.-Aminoisobutylic Acid Using p- and m-(Amidinomethyl)phenyl Esters as Acyl Donor

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