α-Chymotrypsin as the catalyst for peptide synthesis

Morihara, Kazuyuki; Oka, Tatsushi

doi:10.1042/bj1630531

Cited by 126 publications

(28 citation statements)

References 17 publications

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“…The rate constant for ligation varies 8-fold in going from GA to GL to GF showing a mild preference for ligating peptides containing a large hydrophobic residue at P i . This is consistent with data showing a similar preference for hydrolyzing substrates containing large hydrophobic side chains at P i (Morihara et al, 1970). However, for some dipeptide combinations the difference can be much larger; for example, Arg-Phe ligates nearly 100-fold faster than Arg-Gly (showing the importance of the P2' Phe interaction for favorable rate with Arg PI').…”

Section: A) To Sy Of Cys221 Than To the Oy Of Ser221 (492 A)supporting

confidence: 90%

“…A similar trend is observed for extending the peptide substrate from PI' to P,' for hydrolysis (Morihara et al, 1970;Morihara & Oka, 1977). Moreover, the specificity determinants for both reactions are similar from PI' to P; [notably a preference for a larger hydrophobic side chain at P2'; Table V and Morihara et al (1970)l. Taken together, these data suggest that the substrate-binding determinants in the S221C/P225A mutant for ligation are essentially the same as those in wild-type subtilisin for hydrolysis.…”

Section: N F I B Itorsupporting

confidence: 68%

“…CPhilipp and Bender (1983);Carter et al (1989). dInferred from inspection of X-ray structures (Robertus et al, 1972;McPhalen & James, 1988) and from Morihara et al (1970). CCarter and Wells (1987).…”

Section: N F I B Itormentioning

confidence: 99%

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Engineering subtilisin and its substrates for efficient ligation of peptide bonds in aqueous solution

Abrahmsén¹,

Tom²,

Burnier³

et al. 1991

Biochemistry

246

230

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Protein engineering techniques were used to construct a derivative of the serine protease subtilisin that ligates peptides efficiently in water. The subtilisin double mutant in which the catalytic Ser221 was converted to Cys (S221C) and Pro225 converted to Ala (P225A) has 10-fold higher peptide ligase activity and at least 100-fold lower amidase activity than the singly mutated thiolsubtilisin (S221C) that was previously shown to have some peptide ligase activity [Nakatsuka, T., Sasaki, T., & Kaiser, E. T. (1987) J. Am. Chem. SOC. 109, 3808-38101. A 1.5-A X-ray crystal structure of an oxidized derivative of the double mutant (S22 1C/P225A) supports the protein design strategy in showing that the P225A mutation partly relieves the steric crowding expected from the S221 C substitution, thus accounting for its improved catalytic efficiency. Stable and synthetically reasonable alkyl ester peptide substrates were prepared that rapidly acylate the S22 1C/P225A enzyme, and aminolysis of the resulting thioacyl-enzyme intermediate by various peptides is strongly preferred over hydrolysis. The efficiency of aminolysis is relatively insensitive to the sequence of the first two residues in the acyl acceptor peptide whose a-amino group attacks the thioacyl-enzyme.To obtain greater flexibility in the choice of coupling sites, a set of three additional peptide ligases were engineered by introducing mutations into the parent ligase (S22 1 C/P225A) that were previously shown to change the specificity of subtilisin for the residue nearest the acyl bond (the P, residue). The specificity properties of the parent ligase and derivatives of it paralleled those of wild type and corresponding specificity variants. The set of specific peptide ligases should be useful for blockwise synthesis or semisynthesis of proteins in aqueous solution.C h e m i c a l approaches for synthesis and engineering of proteins offer many advantages to recombinant methods in that one can incorporate nonnatural or selectively labeled amino acids. However, peptide synthesis is practically limited to small proteins (typically <50 residues) due to the accumulation of side products and racemization that complicate product purification and decrease yields [for recent reviews see Kaiser (1989) and Offord (1987)l.Proteolytic enzymes, in particular serine proteases, have been used as alternatives to synthetic peptide chemistry because of their stereoselective properties and mild reaction conditions [for reviews see Kullmann (1987) and Chaiken (1981)l. Such enzymes have also been used to complement chemical coupling methods and allow larger proteins to be synthesized by blockwise enzymatic coupling of synthetic fragments [Tnouye et al., 1979; for review see Chaiken (1981)l. However, the narrow substrate specificities and hydrolytic activities of serine proteases have limited their use in peptide synthesis.A central problem in the use of serine proteases for peptide synthesis is that hydrolysis of the acyl-enzyme intermediate is strongly favored over aminoly...

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Section: A) To Sy Of Cys221 Than To the Oy Of Ser221 (492 A)supporting

confidence: 90%

Section: N F I B Itorsupporting

confidence: 68%

See 1 more Smart Citation

Engineering subtilisin and its substrates for efficient ligation of peptide bonds in aqueous solution

Abrahmsén¹,

Tom²,

Burnier³

et al. 1991

Biochemistry

246

230

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“…Serine and sulfhydryl endopeptidases do not catalyze reaction I in Scheme 8 but they do catalyze reactions II and IIIa (136,149) and may The results are from ref. 34.…”

Section: Use Of Serine Carboxypeptidases In Peptide Synthesismentioning

confidence: 89%

“…The structures of the acyl and amine components which may participate in the reaction are determined by the specificity of the enzyme used, and studies in this laboratory have revealed that serine carboxypeptidases, e.g. carboxypeptidase Y and the malt carboxypeptidases, as expected, catalyze only aminolysis reactions with amino acids and amino acid derivatives as amine components (33,38,196,197) as opposed to endopeptidases that in addition to amino acid derivatives also accept polypeptides as nucleophiles (136,149). Consequently, when a carboxypeptidase is used a peptide chain will only be elongated by a single amino acid residue in each aminolysis reaction.…”

Section: Use Of Serine Carboxypeptidases In Peptide Synthesismentioning

confidence: 99%

Serine carboxypeptidases. A review

Breddam

1986

Carlsberg Res. Commun.

180

130

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Carboxypeptidases are proteolytic enzymes which only cleave the C-terminal peptide bond in polypeptides. Those characterized until now can, dependent on their catalytic mechanism, be classified as either metallo carboxypeptidases or as serine carboxypeptidases. Enzymes from the latter group are found in the vacuoles of higher plants and fungi and in the lysosomes of animal cells. Many fungi, in addition, excrete serine carboxypeptidases. Apparently, bacteria do not employ this group of enzymes.Most serine carboxypeptidases presumably participate in the intracellular turnover of proteins and some of them, in addition, release amino acids from extracellular proteins and peptides. However, prolyl carboxypeptidase which cleaves the C-terminal peptide bond of angiotensin II and III is a serine carboxypeptidase with a more specific function.Serine carboxypeptidases are usually glycoproteins with subunit molecular weights of 40,000 -75,000. Those isolated from fungi apparently contain only a single peptide chain while those isolated from higher plants and animals in most cases contain two peptide chains linked by disulfide bridges. However, a number of the enzymes aggregate forming dimers and oligomers.It is probable that the well-known catalytic mechanism of the serine endopeptidases is also employed by the serine carboxypeptidases but presumably with the difference that the plQ of the catalytically essential histidyl residue is somewhat lower in the carboxypeptidases than in the endopeptidases. However, the leaving group specificity of these two groups of enzymes differ since the carboxypeptidases only release C-terminal amino acids from peptides (peptidase activity) and not longer peptide fragments. In addition, they release C-terminal amino acid amides (peptidyl amino acid amide hydrolase activity) or ammonia (amidase activity) from peptide amides and alcohols from peptide esters (esterase activity) and this property they share with the serine endopeptidases. Like other proteolytic enzymes the serine carboxypeptidases contain binding sites which secure the interaction between enzyme and substrate. In this laboratory, the properties of these have been studied for three serine carboxypeptidases, i.e. carboxypeptidase Y from yeast and malt carboxypeptidases I and II, by means of kinetic studies, chemical modifications of amino acid side-chains located at these binding sites and exchange of such amino acid residues by site-directed mutagenesis.Serine carboxypeptidases, such as carboxypeptidase Y and malt carboxypeptidase II which are available in large quantities, can be applied for several purposes. Their broad specificity and ability to release amino acids from the C-terminus of a peptide chain can be employed in determination of amino acid sequences, and their ability to catalyze transpeptidation reactions and aminolysis ofpeptide esters can be employed to exchange C-terminal amino acid residues in peptides and in step-wise synthesis ofpolypeptides, respectively. The type of reactions catalyzed by these enzymes is l...

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Proteinase‐Catalyzed Synthesis of Peptide Bonds

Fruton

1982

Advances in Enzymology - And Related Areas of Molecular Biology

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α-Chymotrypsin as the catalyst for peptide synthesis

Cited by 126 publications

References 17 publications

Engineering subtilisin and its substrates for efficient ligation of peptide bonds in aqueous solution

Engineering subtilisin and its substrates for efficient ligation of peptide bonds in aqueous solution

Serine carboxypeptidases. A review

Proteinase‐Catalyzed Synthesis of Peptide Bonds

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