Carboxypeptidase of Streptomyces griseus. Implications of its characteristics

Breddam, Klaus; Bazzone, Thomas J.; Holmquist, Barton; Vallée, Bert L.

doi:10.1021/bi00575a028

Cited by 36 publications

(13 citation statements)

References 39 publications

(31 reference statements)

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“…However, carboxypeptidase B (1) releases hydrophobic amino acids at very low rates compared to malt carboxypeptidase II and the analogy is thus not perfect. The specificity of malt carboxypeptidase II is more similar to that of the metallo carboxypeptidase from S. griseus which releases both basic and hydropho-bic amino acids at high rates (5).…”

Section: Discussionmentioning

confidence: 87%

Isolation of carboxypeptidase II from malted barley by affinity chromatography

Breddam

Sørensen

Ottesen

1985

Carlsberg Res. Commun.

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A serine carboxypeptidase isolated from malted barley by affinity chromatography was termed malt carboxypeptidase II to distinguish it from another malt carboxypeptidase previously described (Carlsberg Res. Commun. 48, 217-230 (1983)), henceforth called malt carboxypeptidase I. Our nomenclature is in agreement with the nomenclature formerly suggested by MIKOLA. Malt carboxypeptidase II has a molecular weight of 110,000-120,000. It appears to be a dimer where each monomer is composed of two peptide chains linked by disulfide bridges: one monomer contains an A-chain (34,000) and a B-chain (27,000), the other an A-chain and a C-chain (24,000). The enzyme contains 28 residues ofglucosamine and 15% neutral sugar. The N-terminal sequence of the A-chain was NHe-Ala-Gly-Gly-His-Ala-Ala-Asp-Arg-Ile-Val-while the B-and C-chains appeared to be N-terminally blocked. The amino acid compositions of the B-and C-chains were identical suggesting that their different molecular weights are due to different contents of carbohydrate.Malt carboxypeptidase II is inhibited by diisopropyl phosphorofluoridate and by Hg § It exhibits a strong preference for substrates containing Lys and Arg as C-terminal amino acid residues but it also hydrolyses substrates with hydrophobic amino acid residues in this position.

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Section: Discussionmentioning

confidence: 87%

Isolation of carboxypeptidase II from malted barley by affinity chromatography

Breddam

Sørensen

Ottesen

1985

Carlsberg Res. Commun.

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“…22) since the D,D-carboxypeptidases from various strains of bacteria which contain an essential serine residue (193,201) incorrectly have been classified as carboxypeptidases: in addition to cleaving offa C-terminal amino acid residue from a peptide these enzymes also catalyze transpeptidation reactions with nucleophiles containing several amino acid residues, e.g. pentapeptides (164), and this is in conflict with the nucleophile specificity of the serine carboxypeptidases which is limited to single amino acid residues (see section 6.3).…”

Section: Distribution and Function Of Serine Carboxypeptidasesmentioning

confidence: 99%

Serine carboxypeptidases. A review

Breddam

1986

Carlsberg Res. Commun.

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179

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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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“…Carboxypeptidase T (CPT) isolated from Thermouctinomyce,, vulgaris (Osterman et al, 1984) is a remote homologue of mammalian metallocdrboxypeptidases and is similar to carboxypeptidase from Streptomyes griseus (CPSG; Breddam et al, 1979). The CPT molecule consists of a single polypeptide chain of 326 amino acids and contains one zinc ion (Osterman et al, 1984).…”

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Crystal structure of carboxypeptidase T from Thermoactinomyces vulgaris

Teplyakov

Polyakov

Obmolova

et al. 1992

European Journal of Biochemistry

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The crystal structure of carboxypeptidase T from Thermoactirzomyces vulgaris has been determined at 0.235-nm resolution by X-ray diffraction. Carboxypeptidase T is a remote homologue of mammalian Zn-carboxypeptidases. In spite of the low degree of amino acid sequence identity, the threedimensional structure of carboxypeptidase T is very similar to that of pancreatic carboxypeptidases A and B. The core of the protein molecule is formed by an eight-stranded mixed p sheet. The active site is located at the C-edge of the central (parallel) part of the B sheet. The structural organization of the active centre appears to be essentially the same in the three carboxypeptidases. Amino acid residues directly involved in catalysis and binding of the C-terminal carboxyl of a substrate are strictly conserved. This suggests that the catalytic mechanism proposed for the pancreatic enzymes is applicable to carboxypeptidase T and to the whole family of Zn-carboxypeptidases. Comparison of the amino acid replacements at the primary specificity pocket of carboxypeptidases A, B and T provides an explanation of the unusual 'A+B' type of specificity of carboxypeptidase T. Four calcium-binding sites localized in the crystal structure of carboxypeptidase T could account for the high thermostability of the protein.Metallocarboxypeptidases are exopeptidases which contain a zinc ion in the active centre and catalyze the hydrolysis of C-terminal amino acids from polypeptide substrates. Extensive studies of bovine pancreatic carboxypeptidase A (CPA) by different techniques including crystallography, spectroscopy, kinetics and site-directed mutagenesis resulted in the understanding of the general principles of the catalytic mechanism (Christianson and Lipscomb, 1989). Three-dimensional structures have been determined for pancreatic carboxypeptidases A (Rees et al., 1983;Kobe and Goldsmith, 1990) and B (Schmid and Herriott, 1976; Coll et al., 1991) which share a common polypeptide fold but differ in substrate specificity (Hartsuck and Lipscomb, 1971 ;Folk, 1971 polypeptide chain of 326 amino acids and contains one zinc ion (Osterman et al., 1984). The amino acid sequence of CPT, deduced from the gene encoding the protein (Smulevitch et al., 1991), shows only moderate similarity with the CPA and CPB sequences; 30% and 27% identical residues, respectively. CPT exhibits an unusual 'dual'-substrate specificity combining features of both CPA and CPB; it is able to split off hydrophobic and basic amino acids with comparable efficiency (Osterman et al., 1984). As other proteins from Thermoactinomyces, CPT has an increased thermal stability in the presence of calcium ions (Osterman et al., 1984).Structure/functional studies of CPT provide a possibility to study the general principles of catalysis by Zn-carboxypeptidases. On the basis of the comparative primary-structure analysis, all Zn-carboxypeptidases were divided into two groups ; ,,digestive,, and ,,regulatory,, enzymes (Osterman et al., 1992). Pancreatic CPA and CPB appear to be structurally ...

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Carboxypeptidase of Streptomyces griseus. Implications of its characteristics

Cited by 36 publications

References 39 publications

Isolation of carboxypeptidase II from malted barley by affinity chromatography

Isolation of carboxypeptidase II from malted barley by affinity chromatography

Serine carboxypeptidases. A review

Crystal structure of carboxypeptidase T from Thermoactinomyces vulgaris

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