Isolation of a carboxypeptidase from malted barley by affinity chromatography

Ottesen

1983

Carlsberg Res. Commun.

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Malt carboxypeptidase (Carlsberg Res. Commun. 48, 217-230, 1983) catalyzes hydrolysis of ester substrates with the general formula Bz-X-OMe with preference for substrates where X = Phe, Arg or Lys over those where the X-position is occupied by an amino acid residue with a neutral or acid aliphatic side chain. The rapid hydrolysis of Bz-Phe-OMe is mainly due to a high k~, value while for Bz-Arg-OMe it is mainly due to a low Km value. Addition of salts result in decreased rates of hydrolysis of substrates where X = Lys or Arg (positively charged) and increased rates of hydrolysis of substrates where X = Phe or Leu (hydrophobic).Guanido compounds influence the enzymatic properties of malt carboxypeptidase. Kinetic data for the hydrolysis of various substrates indicate that phenylguanidine binds to the St' binding site of the enzyme in such a manner that the rate of cleavage of ester or amide substrates with large groups in the P( position, i.e. -OEt, -OPt, -OBu, -Ala-OH, -Gly-NH2, is reduced while substrates with small groups in this position i.e. -OMe, -NH2 are hydrolyzed with increased rates. These observations have applications in deamidation reactions since the presence of phenylguanidine in the reaction medium results in a significantly increased yield of the deamidated product.Abbreviations: Ac = acetyl; BiGu = 2-guanidobenzimidazol; Bu = butyl; Bz = benzoyl; DFP = diisopropylphosphorofluoridate; EDTA = ethylene diamine tetraacetic acid sodium salt; FA = furylacryloyl; Gu = guanidine hydrochloride; Hepes = N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid; HPLC = high performance liquid chromatography; MeGu = methyl guanidine hydrochloride; Mes = 2-(N-morpholino)ethane sulfonic acid; PhGu = phenylguanidine hydrogencarbonate; PpGu = l-(3-phenylpropylamino) guanidine hydrochloride; Pr = propyl; TEAP = triethyl ammonium phosphate; "Iris = tris(hydroxy methyl) aminomethane; Z = carbobenzoxy. Abbreviations of amino acids, amino acid derivatives and peptides are according to the guidelines of the IUPAC-IUB Commision on Biochemical Nomenclature. The binding notation for the enzyme and the substrates is that of SCHECHTER and BERGER. Accordingly, amino acid residues in the substrate are referred to as P,,P2....P~ in the aminoterminal direction away from the scissible bond and P~ for the carboxy-terminal residue that is hydrolyzed off.

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Influence of guanidine derivatives on the specificity of malt carboxypeptidase

Ottesen

1983

Carlsberg Res. Commun.

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“…However, it has not been established whether these carboxypeptidases represent isoenzymes. The carboxypeptidases isolated from germinated barley grains exhibit a similar complexity (11,28,37,135,154,190,200).…”

Section: Serine Earboxypeptidases In Higher Plantsmentioning

confidence: 99%

“…The amino acid composition of several serine carboxypeptidases has been determined (28,37,79,91,98,110,111,114,188,191) and it is characteristic that the enzymes isolated from fungi have much higher contents of the amino acids Asx and Glx and lower contents of the basic amino acids, i.e. Lys and Arg, than those from higher plants.…”

Section: Physico-chemical Properties Of Serine Carboxypeptidasesmentioning

confidence: 99%

Serine carboxypeptidases. A review

1986

Carlsberg Res. Commun.

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179

130

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...

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

Mortensen

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.