Identification of methionyl and cysteinyl residues in the substrate binding site of carboxypeptidase Y

Breddam, Klaus; Svendsen, Ib

doi:10.1007/bf02907496

Cited by 28 publications

(22 citation statements)

References 21 publications

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“…All the enzymes contain disulfide bridges, and the enzymes from yeast (6,27) penicillum (79) and carboxypeptidase I from barley (28) have been shown in addition to contain a single buried sulfhydryl group (further details are given in section 6). Approximately 98% of the amino acid sequence ofcarboxypeptidase Y has been identified by SVENDSEN and coworkers (124,179) utilizing traditional Edman degradation, and DNA sequencing has recently completed the primary structure (T. STEVENS, personal communications) (36,176). At present, approximately 80% of the amino acid sequences of the A-chains of malt carboxypeptidases I and II are known and they indicate 35% homology with each other and approximately 30% homology with the N-terminal portion of yeast carboxypeptidase (S. SORENSEN, K. BREDDAM, I. SVENDSEN and M. OTTESEN, unpublished observations) suggesting that the serine carboxypeptidases from higher plants and those from fungi are related.…”

Section: Physico-chemical Properties Of Serine Carboxypeptidasesmentioning

confidence: 99%

“…Carboxypeptidase Y contains four carbohydrate moieties which are attached to the protein through asparagine side chains (65,183) at positions tentatively identified by amino acid sequencing (36). All four positions are characterized by an Asn-X-Thr/ Ser sequence which is typical in glycoproteins with N-linked carbohydrates (85).…”

Section: Physico-chemical Properties Of Serine Carboxypeptidasesmentioning

confidence: 99%

“…It has long been known that carboxypeptidase Y from baker's yeast contains a cysteinyl residue (71) and this has recently, in this laboratory, been identified in the amino acid sequence as Cys-341 (36). This residue is inaccessible to iodoacetate and EUman's reagent unless the enzyme is denatured.…”

Section: Modifications At the S Binding Sitementioning

confidence: 99%

“…While the metallo carboxypeptidases are wellcharacterized both with respect to phylogenetic relations and catalytic mechanism, much less is known about the serine carboxypeptidases and only for one of the enzymes, carboxypeptidase Y from yeast, is the primary structure known (36). However, the interest for these enzymes has increased in recent years as evidenced by the number of publications concerning their cellular location, physiological function, structure, mechanism of action, and applications in sequence determination and enzymatic peptide synthesis.…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Physico-chemical Properties Of Serine Carboxypeptidasesmentioning

confidence: 99%

Section: Physico-chemical Properties Of Serine Carboxypeptidasesmentioning

confidence: 99%

Section: Modifications At the S Binding Sitementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Serine carboxypeptidases. A review

Breddam

1986

Carlsberg Res. Commun.

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180

130

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“…Since the amino acid occupying position 398 is part of the S~ binding site (8,9) Cys-CPD-Y was characterized kineticaUy using a series of FA-ester, FA-peptide and FA-amide substrates with different groups in the P; position. Exchange of Met-398 with a cysteinyl residue resulted in a reduction in k~,dK~ for all the substrates listed in Table I and [I (with the exception of FA-Ala-OBzl) due to decreased k~, values and increased Km values.…”

Section: Peptide Synthesis and Deamidation Of Peptide Amidesmentioning

confidence: 99%

Chemical modifications of a cysteinyl residue introduced in the binding site of carboxypeptidase Y by site-directed mutagenesis

Bech

Breddam

1988

Carlsberg Res. Commun.

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It is demonstrated that site-directed mutagenesis successfully can be combined with chemical modification creating enzyme derivatives with altered properties. A methionyl residue located in the S] binding site of carboxypeptidase Y was replaced by a cysteinyl residue and the mutant enzyme was isolated and modified with various alkylating and thioalkylating reagents. Treatment of the mutant carboxypeptidase Y with bulky reagents like phenacyl bromide and benzyl methanethiolsulfonate caused a drastic reduction in the activity towards substrates with bulky leaving groups in the P] position, i.e. -OBzl, -VaI-NH2 and amino acids (except -Gly-OH), while substrates with small groups in that position, i.e. -OMe and -NH2, were hydrolysed with increased rates. The presence of a positive charge, in addition to a bulky group, had a further adverse effect on the activity towards substrates with large leaving groups, whereas the activity towards those with small leafing groups remained unaffected by such a group. The derivatives obtained by modification of the mutant enzyme with benzyl methanethiolsuifonate and methyl methanethiolsulfonate were effective in deamidations of peptide amides and peptide synthesis reactions, respectively. SCHECHTER and BERGER (17). Accordingly, the binding site for the C-terminal amino acid residue is denoted S~ and those for the amino acid residues in the amino-terminal direction away from the scissile bond are denoted S~,S2,...,S,. Amino acids in the substrate are referred to as Pt,P2,-..,P. and P~ in correspondence with the binding sites. Springer-Verlag

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Identification of the catalytic histidine residue participating in the charge‐relay system of carboxypeptidase Y

1995

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Abstract:The essential histidine residue of carboxypeptidase Y (CPY) was modified by a site-specific reagent, a chloromethylketone derivative of benzyloxycarbonyl-L-phenylalanine. The single modified histidine residue was converted to N' -carboxymethyl histidine (cmHis) upon performic acid oxidation. A peptide containing cmHis was isolated from the tryptic-thermolytic digest. Based on the amino acid composition and sequence analysis, the peptide is shown to be Val-Phe-Asp-Gly-Gly-cmHisMet0,-Val-Pro, which was derived from CPY cleaved by trypsin at Arg 391 and thermolysin at Phe 401, and thus His 397 was modified. This histidine residue has been implicated previously by X-ray analysis to participate in the charge-relay system of CPY.

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Identification of methionyl and cysteinyl residues in the substrate binding site of carboxypeptidase Y

Cited by 28 publications

References 21 publications

Serine carboxypeptidases. A review

Serine carboxypeptidases. A review

Chemical modifications of a cysteinyl residue introduced in the binding site of carboxypeptidase Y by site-directed mutagenesis

Identification of the catalytic histidine residue participating in the charge‐relay system of carboxypeptidase Y

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