Peptide bond synthesis by clostridiopeptidase B

Fortier, Guy; MacKenzie, S. L.

doi:10.1007/bf01020821

Cited by 13 publications

(3 citation statements)

References 13 publications

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“…In this case, partition values comprise the sum for the two products (Po = 8.84k0.23). The second peak probably belongs to a transpeptidation product which was observed by Fortier and MacKenzie (1986a) and Yagisawa et al (1990).…”

Section: Discussionmentioning

confidence: 99%

The specificity of clostripain from Clostridium histolyticum

Ullmann¹,

Jakubke²

1994

European Journal of Biochemistry

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The S' subsite specificity of clostripain from Clostridium histolyticum was investigated by acyl transfer to libraries of amino acid amides, Ala-Xaa dipeptides, proline derivatives and pentapeptides using Nu-benzoyl-L-arginine ethyl ester as acyl donor. A pentapeptide library consisting of 29 pentapeptides with general structure Xaa-Ala-Ala-Ala-Gly, Ala-Xaa-Ala-Ala-Gly and Ala-Ala-XaaAla-Gly, where Xaa represents Gly, Ala, Pro, Leu, Phe, Asp, Glu, Arg and Lys, was prepared by solid-phase synthesis. The data analysis was performed by HPLC and evaluated by statistical algorithms. The nucleophile efficiency covers a range of more than three orders of magnitude. In the P; position, low specificity for amino acid amides and Xaa-(Ala),-Gly peptides was found, however, in the Pi position, positively charged amino acid residues are strongly preferred. The negatively charged side chains of aspartic acid and glutamic acid in the P: and Pi positions, respectively, show only poor nucleophilic behaviour. In the case of these amino acids, the S'-P' interactions depend significantly on their position of these residues in the peptide chain of the nucleophile. The transfer of aspartic acid and glutamic acid from P; -Pi increases the nucleophile efficiency by approximately two orders of magnitude. The aromatic side chains are not well accepted, especially in the case of PiPhe. Surprisingly, P:Gly leads to effective P'-S' interactions. However, the opposite result was obtained for PiGly. The high efficiency for Gly-NH, does not fit with the hydrophobicity structure/ activity relationships. In most cases, peptide chain elongation does not improve the nucleophile efficiency. The effective interaction of D-Leu-NH, with the S' subsite of clostripain emphasizes the fact that the nucleophile stereospecificity is not restricted to L-amino acids. The results with proline derivatives indicate remarkably different specificities of the S' binding site which can only be explained by conformational restraints. A positive cooperativity between P:Pro and PiGly and a negative cooperativity between Pi Pro and PiPhe was observed. The arrangement of three proline residues next to each other represents a favourable conformation for effective enzyme-nucleophile interactions.The cysteine protease clostripain (Clostridium histolyticum proteinase B) from the culture filtrate of C. histolyticum is well known for its restricted substrate specificity, the cleavage of Arg-Xaa bonds, including proline in the P; position (subsite nomenclature of Schechter and Berger, 1967) (Ogle andTytell, 1953 ;Labouesse and Gros, 1960; Mitchell, 1964). For this reason this enzyme is important both in sequence analysis and in enzymic peptide synthesis, because proline is normally not well accepted in the P: position by the majority of proteases. Furthermore, a precise characterization of clostripain specificity is important if selective inhibitors are to be designed.Although S subsites have been studied in detail (Cole et al., 1971;Porter et al., 1971;Gilles et al., 1979;

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Section: Discussionmentioning

confidence: 99%

The specificity of clostripain from Clostridium histolyticum

Ullmann¹,

Jakubke²

1994

European Journal of Biochemistry

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“…are given in Table 2. were carried out at about pH 9.2, because the deprotonated form of the amine components was assumed to be the reactive species in the reaction from the pHdependence of the transpeptidation between Bz-Arg-OEt and leucinamide, which was represented by a normal titration curve with a middle point at pH 7.6. (The pH-yield profiles obtained at a constant reaction time showed a pH optimum [12], but this must be a result of the low enzyme activity at high pH.) Fig.…”

Section: Resultsmentioning

confidence: 90%

“…Polymerization of dipeptide amides and amino acid esters by cathepsin C [8] and papain [9] suggests a high efficiency of cysteine proteinases in transpeptidation at low substrate concentrations. Clostripain has been used in the condensation of protein fragments by means of a reverse reaction [3,4,10] and in the preparation of oligopeptides by means of a transpeptidation [11,12]. However, high efficiency of transpeptidation at low substrate concentrations has not been demonstrated conclusively or utilized effectively.…”

Section: Introductionmentioning

confidence: 99%

High-efficiency transpeptidation catalysed by clostripain and electrostatic effects in substrate specificity

Yagisawa

Watanabe

Takaoka

et al. 1990

Biochemical Journal

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Clostripain catalyses the transpeptidation between benzoylarginin ethyl ester and amino acid amides, oligopeptides, insulin A- and B-chains and tryptic peptides of myoglobin at millimolar substrate concentrations. The reactions proceed with temporary accumulation of the products, followed by hydrolytic decomposition. The yield was not affected significantly by the type of N-terminal amino acid, but was diminished markedly by the negative charges of the amine components. The yields for natural peptides were linearly related to the charge density of the peptides.

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Proteasekatalysierte kinetisch kontrollierte Peptidsynthese

Schellenberger¹,

Jakubke²

1991

Angewandte Chemie

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Peptide bond synthesis by clostridiopeptidase B

Cited by 13 publications

References 13 publications

The specificity of clostripain from Clostridium histolyticum

The specificity of clostripain from Clostridium histolyticum

High-efficiency transpeptidation catalysed by clostripain and electrostatic effects in substrate specificity

Proteasekatalysierte kinetisch kontrollierte Peptidsynthese

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