Primary structure of carboxypeptidase I from malted barley

Sørensen, Steen Bech; Breddam, Klaus; Svendsen, Ib

doi:10.1007/bf02906889

Cited by 47 publications

(36 citation statements)

References 23 publications

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“…Earlier investigations (6) have shown that malt carboxypeptidase II, like wheat carboxypeptidase II (7) and malt carboxypeptidase I (5,18), is composed of two subunits where each subunit is constituted of two peptide chains, an A-chain and a B-chain, linked together by disulphide bridges. Determination of the amino acid sequence of serine carboxypeptidase II from barley malt revealed that the A-chain contained 260 amino acid residues ( Figure 9).…”

Section: Discussionmentioning

confidence: 99%

“…Chemical cleavage by cyanogen bromide and hydroxylamine and enzymatic digestion with trypsin of the reduced and alkylated chains was performed as previously described (18).…”

Section: Cleavage Of Peptide Bondsmentioning

confidence: 99%

“…These enzymes in combination with endopeptidases, very effectively produce free amino acids during germination by cleavage of reserve proteins in the endosperm. We have previously reported the sequence of malt carboxypeptidase I (18). This enzyme consists, like a number of serine carboxypeptidases from higher plants (2), of two identical subunits, each composed of two peptide chains, cross-linked by disulphide bridges (6).…”

Section: Introductionmentioning

confidence: 99%

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Primary structure of carboxypeptidase II from malted barley

Sørensen¹,

Svendsen²,

Breddam³

1987

Carlsberg Res. Commun.

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The primary structure of malt carboxypeptidase II has been determined. The enzyme is a dimer where each monomer is composed of two peptide chains, an A-and a B-chain, linked by disulphide bridges. The B-chain exists in two forms, both N-terminally blocked, which differ in position 38 and 39, one form containing Ala3S-Thr 39, the other containing Thr38-Asn 39 with carbohydrate attached to the asparagine side-chain. Fragments of the A-and B-chains were obtained by chemical cleavages with either cyanogen bromide, hydroxylamine or iodosobenzoic acid and by enzymatic cleavages with either trypsin or S. aureus V8 protease, sequenced and aligned to give the total sequence.The A-and B-chains contain 260 and 159 amino acid residues, respectively. Glycosylated asparagines are found in positions 114, 125 and 257 of the A-chain, and positions 28, 34, 39 and 159 of the B-chain. Position 39 of the B-chain is only partially glycosylated (see above). Alignment of the sequence of the A-chain with the N-terminal part of carboxypeptidase Y revealed 28% homology. Similarly, the B-chain showed 21% homology with the C-terminal part of carboxypeptidase Y. The homology between malt carboxypeptidases I and II is 40% for the A-chains and 30% for the B-chains. The corresponding homologies between malt carboxypeptidase II and wheat carboxypeptidase II are 95% and 96%, respectively. No homology was observed with other proteins by a computer search of a sequence data base provided by the National Biomedical Research Foundation. A region of the A-chain was identical to the region around the essential seryl residue in position 146 of carboxypeptidase u

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Section: Discussionmentioning

confidence: 99%

“…Chemical cleavage by cyanogen bromide and hydroxylamine and enzymatic digestion with trypsin of the reduced and alkylated chains was performed as previously described (18).…”

Section: Cleavage Of Peptide Bondsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Primary structure of carboxypeptidase II from malted barley

Sørensen¹,

Svendsen²,

Breddam³

1987

Carlsberg Res. Commun.

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“…However, CPD-Mlll is a monomeric enzyme, consisting of a single peptide chain while malt carboxypeptidases I and II are dimeric with each monomer consisting of two peptide chains, linked by disulfide bridges (2,6,23,24). Furthermore, CPD-M.~ contains far less carbohydrate than the other two carboxypeptidases.…”

Section: Discussionmentioning

confidence: 99%

“…The serine carboxypeptidases play an important role in this process and MmOLA ( 17,18,19) has presented evidence for the presence in malt of five such enzymes with complementary specificites. In this laboratory two of these have been isolated by affinity chromatography and their enzymatic properties and sequences have been determined (2,3,4,6,7,23,24). The isolation and characterization of CPD-Mm is reported here.…”

Section: Introductionmentioning

confidence: 99%

Isolation of carboxypeptidase III from malted barley by affinity chromatography

Breddam

Sørensen

1987

Carlsberg Res. Commun.

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A novel serine carboxypeptidase has been isolated from malted barley by affinity chromatography. The enzyme has been iermed malt carboxypeptidase III in correspondence with the nomenclature suggested by MIKOLA to distinguish it from the previously isolated carboxypeptidases I and II. Malt carboxypeptidase III is a monomer with a molecular weight around 48,000, composed of a single peptide chain, two glucosamine residues and 2% neutral sugar. The peptide chain is N-terminally blocked and it contains a single unblocked cysteinyl residue.Like other serine carboxypeptidases malt carboxypeptidase III exhibits peptidase activity with an acidic pH optimum. However, unlike other such enzymes, the pH optimum for the esterase activity is also acidic. The kinetic analysis showed that this probably is due to deprotonation of an ionizable group with a pI~ around 7 which controls the conformation of the active site. The transition of the active acidic form into the inactive basic form is reversible but time dependent.Malt carboxypeptidase III exhibits a preference for substrates with Phe, Met, Ile and Val in the P] position and Phe, Leu, Met and Ala in the P~ position. The enzyme is inhibited by diisopropyl phosphorofluoridate, mercuric ions and various organomercuric compounds.

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A conserved glutamic acid bridge in serine carboxypeptidases, belonging to the α/β hydrolase fold, acts as a pH‐dependent protein‐stabilizing element

Mortensen

Breddam

1994

Protein Science

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Serine endopeptidases of the chymotrypsin family contain a salt bridge situated centrally within the active site, the acidic component of the salt bridge being adjacent to the catalytically essential serine. Serine carboxypeptidases also contain an acidic residue in this position but it interacts through a short hydrogen bond, probably of low‐barrier type, with another acidic residue, hence forming a “glutamic acid bridge.” In this study, the residues constituting this structural element in carboxypeptidase Y have been replaced by site‐specific mutagenesis. It is demonstrated that the glutamic acid bridge contributes significantly to the stability of the enzyme below pH 6.5 and has an adverse effect at pH 9.5. Carboxypeptidase WII from wheat contains 2 such bridges, and it is more stable than carboxypeptidase Y at acidic pH.

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Primary structure of carboxypeptidase I from malted barley

Cited by 47 publications

References 23 publications

Primary structure of carboxypeptidase II from malted barley

Primary structure of carboxypeptidase II from malted barley

Isolation of carboxypeptidase III from malted barley by affinity chromatography

A conserved glutamic acid bridge in serine carboxypeptidases, belonging to the α/β hydrolase fold, acts as a pH‐dependent protein‐stabilizing element

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