Influence of the substrate structure on carboxypeptidase Y catalyzed peptide bond formation

Breddam, Klaus; Widmer, Fred; Johansen, Jeppe

doi:10.1007/bf02906160

Cited by 29 publications

(24 citation statements)

References 18 publications

(12 reference statements)

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“…CPD-Y, N-benzoyl-glycine methyl ester, Nbenzoyl-L-alanine benzyl ester and N-benzoyl-Lphenylalanine methyl ester were all prepared as previously described (3,6). N-benzoyl-L-lysyl-Lalanine was prepared by enzymatic synthesis (to be published).…”

Section: Methodsmentioning

confidence: 99%

Modification of the single sulfhydryl group of carboxypeptidase Y with mercurials. Influence on enzyme specificity

Breddam

1983

Carlsberg Res. Commun.

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Modification of the single sulthydryl group of carboxypeptidase Y with one equivalent Hg -~ resulted in an inactive enzyme when assayed in the absence ofhalides regardless of the nature of the amino acid residue in the P, position of the ester substrate. Anions capable ofcomplexing with Hg ~, i.e., CI-, Br-, I-, SCN-and CN-, partially reactivated the Hg ~ modified CPD-Y to a level of 20-28% of the esterase activity of the unmodified enzyme. The concentration of anion necessary to achieve maximal activation varied and the following dissociation constants were determined: CI-: pK~ = 1.8, SCN-: pK~ = 3.4, Br-: pl~ = 3.6, I-: pK~ = 5.4, CN-: pK d > 7. The relation between activation and concentration of anion was in agreement with binding of one equivalent of anion to the enzyme bound mercury. A similar reactivation was not observed for CPD-Y partially inactivated with phenylmercuric chloride.The kinetic constants for a series of ester substrates with the general formula Bz-X-OMe, where X = amino acid residue, were determined for unmodified CPD-Y, CPD-Y modified with Hg ~ (assayed in the presence of I-) and CPD-Y reacted with phenylmercuric chloride. Unmodified CPD-Y exhibited a strong preference for substrates with hydrophobic amino acid residues, i.e., Phe, Leu, Met, and only slowly hydrolyzed substrates with hydrophilic amino acid residues, i.e., Arg, Lys, Thr, Gly. The two modified enzymes also had the highest k~JKm for substrates with hydrophobic residues. However, their relative preference for these different substrates deviated substantially from the unmodified enzyme. The increased preference for Bz-Lys-OMe when the Hg ++ modified enzyme was used and for Bz-Gly-OMe and Bz-AIa-OMe when the phenylmercuric chloride modified enzyme was used were the most pronounced changes in the specificity of CPD-Y following its modification with mercurials.

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Section: Methodsmentioning

confidence: 99%

Modification of the single sulfhydryl group of carboxypeptidase Y with mercurials. Influence on enzyme specificity

Breddam

1983

Carlsberg Res. Commun.

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“…to alcohols, amino acids and amino acid derivatives (24,33,38,195,196,197). For carboxypeptidase Y it has been shown, using N-blocked amino acid esters as substrates, that the fraction ofaminolysis, P3/(P2+P3), is highly dependent on the nature of the amine nucleophile added.…”

Section: Is a Linear Function Of The Ratio [N]/[w]mentioning

confidence: 99%

“…These results demonstrate that the speci- specificity of the enzyme employed imposes severe restrictions on the type of reactions which are feasible and in addition, they demonstrate how chemically modified derivatives of enzymes may be useful. Although the yields of carboxypeptidase Y catalyzed transpeptidation reactions are strongly dependent on the structures of both the acyl and amine components used in the reaction (23,24) they represent attractive possibilities in protein semisynthesis. Exchange of C-terminal amino acid residues in peptides, incorporation of labelled amino acids as well as reporter groups and conversion of peptides to peptide esters are possibilities to be considered.…”

Section: 4mentioning

confidence: 99%

Serine carboxypeptidases. A review

Breddam

1986

Carlsberg Res. Commun.

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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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“…Previous reports have examined the effect of nucleophiles with different carboxyl groups on aminolysis reaction yields using N-protected peptide alkyl esters. 30,31 Among the amino-acid alkyl esters, amino-acid amides and free amino acids, amino-acid alkyl esters were the most suitable substrate for oligomerization because the resulting peptide was obtained directly in an ester form and was ready to react with the enzyme again, leading to consecutive additions of the same nucleophile. Thus, amino-acid methyl esters were chosen as the nucleophiles in this study.…”

Section: Resultsmentioning

confidence: 99%

“…This corresponds to the findings from a previous report that showed that CPDY prefers to react with hydrophobic amino acids as opposed to hydrophilic amino acids. 31 These data indicated that CPDY-catalyzed oligomerization was affected by both the type of amino acid and the size of the ester groups.…”

Section: Type Of Amino Acidsmentioning

confidence: 94%

Synthesis of peptides with narrow molecular weight distributions via exopeptidase-catalyzed aminolysis of hydrophobic amino-acid alkyl esters

Nitta

Komatsu

Ishii

et al. 2016

Polym J

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A new synthesis technique that produces homogeneous oligopeptides that are essential for the formation of self-assembled structures is described. In contrast with endopeptidases, exopeptidases, which catalyze the cleavage of terminal peptide bonds, can potentially prevent unexpected hydrolysis during aminolysis. This is the first report on exopeptidase-catalyzed oligopeptide synthesis. Oligo(L-leucine) was synthesized using the exopeptidase carboxypeptidase Y (CPDY), which also prevented enzymatic hydrolysis. A high yield of oligo(L-leucine) with a narrow polydispersity (PDI) was obtained by limiting polypeptide cleavage during aminolysis. The reaction conditions, such as the temperature, pH, CPDY and substrate concentrations, were optimized to produce the best yields, molecular weights and PDI of the oligomers. Hydrophobic L-leucine methyl ester and L-isoleucine methyl ester were found to be the appropriate substrates for CPDY-catalyzed oligomerization; however, no oligomers were obtained using hydrophilic amino-acid esters. Oligomer yields were also affected by the amino-acid ester groups. Remarkably, the PDI of the oligomers was as low as 1.0, regardless of the reaction conditions and types of substrates used. Thus, exopeptidase-catalyzed oligomerization may become an alternative route for the current endopeptide-catalyzed oligomerization of peptides.

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Influence of the substrate structure on carboxypeptidase Y catalyzed peptide bond formation

Cited by 29 publications

References 18 publications

Modification of the single sulfhydryl group of carboxypeptidase Y with mercurials. Influence on enzyme specificity

Modification of the single sulfhydryl group of carboxypeptidase Y with mercurials. Influence on enzyme specificity

Serine carboxypeptidases. A review

Synthesis of peptides with narrow molecular weight distributions via exopeptidase-catalyzed aminolysis of hydrophobic amino-acid alkyl esters

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