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K Breddam

Publications and source records attributed to K Breddam.

52 records · Page 3Linked to original sources
PubMed1991

Fragmentation of proteins by S. aureus strain V8 protease. Ammonium bicarbonate strongly inhibits the enzyme but does not improve the selectivity for glutamic acid.

Staphylococcus aureus strain V8 protease is a serine endopeptidase which cleaves peptide bonds at the carboxyl side of Glu and Asp. Specific cleavage at Glu has previously been achieved in ammonium bicarbonate whereas in sodium phosphate cleavage at both Glu and Asp was observed. However, it is shown here that bicarbonate does not restrict the specificity to Glu-X bonds, it simply inhibits the enzyme. The degradation of a mixture of oxidized insulin and glucagon proceeds similarly in the two buffers, although faster in phosphate.

Amino Acid Sequence↗
PubMed1991

Anthranilamide and nitrotyrosine as a donor-acceptor pair in internally quenched fluorescent substrates for endopeptidases: multicolumn peptide synthesis of enzyme substrates for subtilisin Carlsberg and pepsin.

The preparations of N alpha-Fmoc-3-nitro-L-tyrosine and N-Boc-anthranilic acid Dhbt ester and their application to parallel multiple column solid-phase peptide synthesis is described. A series of peptide substrates containing an anthraniloyl group at the amino terminus and a 3-nitrotyrosyl residue close to the carboxyl terminus have been synthesized. The fluorescence of the anthraniloyl group, intramolecularly quenched by the 3-nitrotyrosine, increases with cleavage of peptide bonds situated between the two groups. The quenching mechanism is of the long-range resonance energy transfer type and long peptide substrates were constructed and used for kinetic measurement on subtilisin Carlsberg and pepsin. Complete quenching was observed even with more than 20 A between the centers of the chromophores, and substrates with up to 50 A between the chromophores were synthesized. The importance of long substrates for optimal enzymatic activity was demonstrated.

Amino Acid Sequence↗
PubMed1991

Amidation of growth hormone releasing factor (1-29) by serine carboxypeptidase catalysed transpeptidation.

The applicability of serine carboxypeptidase catalysed transpeptidation reactions, using amino acid amides as nucleophiles, for C-terminal amidation of peptides has been investigated. With the aim of converting an unamidated precursor into GRF(1-29)-NH2, an interesting biologically active derivative of growth hormone releasing factor, a number of model reactions were initially investigated. In such a transpeptidation reaction, where the C-terminal amino acid is replaced by the amino acid amide, used as nucleophile, the C-terminal amino acid residue of the substrate can be chosen freely since it functions as leaving group and does not constitute part of the product. Since the C-terminal sequence of GRF(1-29)-NH2 is -Met-Ser-Arg-NH2 the model reactions Bz-Met-Ser-X-OH (X = Ala, Leu, Arg) + H-Arg-NH2----Bz-Met-Ser-Arg-NH2 + H-X-OH were first studied. With carboxypeptidase Y and X = Ala or Leu the amidated product could be obtained of 98% and 41%, respectively. With carboxypeptidase W-II and X = Arg a yield of no more than 72% could be obtained. The choice of Ala as leaving group in combination with carboxypeptidase Y therefore appeared optimal. With the longer peptide Bz-Leu-Gln-Asp-Ile-Met-Ser-Ala-OH the amidated product could be obtained in a yield of 78%, using carboxypeptidase Y, the only other product being Bz-Leu-Gln-Asp-Ile-Met-Ser-OH, formed due to the competing hydrolysis reaction. The full length peptide GRF(1-28)-Ala-OH was synthesized by the continuous flow polyamide solid-phase method.(ABSTRACT TRUNCATED AT 250 WORDS)

Amides↗
PubMed1990

A highly active and oxidation-resistant subtilisin-like enzyme produced by a combination of site-directed mutagenesis and chemical modification.

The subtilisins are known to be susceptible to chemical oxidation due to the conversion of Met222 into the corresponding sulfoxide. A number of derivatives with resistance towards oxidation have previously been prepared by replacement of this group with the other 19 amino acid residues. Unfortunately, the activities of these enzymes were of the order of 1-10% of that obtained with the wild-type enzyme. In contrast, the oxidation-labile cysteine mutant exhibited much higher activity, suggesting that this is associated with the presence of a sulphur atom in the amino acid at position 222. It is shown here that it is possible to maintain a sulphur atom in the amino acid at position 222 without the enzyme becoming labile towards oxidation. A subtilisin from Bacillus lentus, subtilisin 309, in which Met222 was replaced with a cysteinyl residue by site-directed mutagenesis was modified with thioalkylating reagents. Treatment of such enzyme derivatives with H2O2 revealed that their stabilities towards oxidation had increased significantly compared to both wild-type and unmodified [Cys222]subtilisin. One of the chemically modified enzyme derivatives, [Me-S-Cys222]subtilisin, exhibited a kcat/Km value of 56% of that obtained with the wild-type enzyme when assayed against the substrate Suc-Ala-Ala-Pro-Phe-NH-Ph-NO2 (Suc, succinyl) and it exhibited 89% activity when tested in an assay with dimethyl casein as a substrate. The corresponding values obtained for unmodified [Cys222]subtilisin were lower, i.e. 39% for the dimethyl casein activity and 46% for the kcat/Km for the hydrolysis of Suc-Ala-Ala-Pro-Phe-NH-Ph-NO2. This demonstrates the feasibility of replacing the oxidation-labile methionyl residue group in a subtilisin enzyme with a group stable towards oxidation without substantially reducing the activity.

Bacillus↗
PubMed1990

Crystallization of serine carboxypeptidases.

Crystallization of three different serine carboxypeptidases has been achieved by the method of hanging-drop vapor diffusion. Serine carboxypeptidases II from wheat bran and malted barley crystallize isomorphously from polyethylene glycol solutions at room temperature (pH 4 to 7) in space group P4(1)2(1)2 or enantiomorph with cell dimensions of a = b = 98.2 A and c = 209.5 A. The crystals diffract to about 2.3 A resolution using rotating-anode X-ray generators. Assuming a dimer of Mr 120,000 in the asymmetric unit, Vm = 2.1 A3/dalton. These crystals appear suitable for structural studies. A genetically engineered serine carboxypeptidase from yeast, which lacks three of four glycosylation sites present in the wild-type, has also been crystallized by vapor diffusion against methylpentanediol at 4 degrees C, pH 6.4 to 8.0.

Carboxypeptidases↗
PubMed1989

Inactivation of carboxypeptidase Y by mutational removal of the putative essential histidyl residue.

Carboxypeptidase Y is a serine carboxypeptidase assumed to contain a catalytic triad similar to the serine endopeptidases. On the basis of the homology between various serine carboxypeptidases His-397 is suspected to be part of the catalytic triad. To test this it was exchanged with Ala and Arg by site-directed mutagenesis of the cloned PRC1 gene. The catalytic efficiency of the mutant enzymes were reduced by a factor of 2 X 10(4) and 7 X 10(2), respectively, confirming the key role of His-397 in catalysis. Treatment of Ala-397-CPD-Y with Hg++ or CNBr, hence modifying Cys-341 located in the vicinity of the active site abolished the residual activity of the enzyme, indicating an additional involvement of this residue in catalysis.

Amino Acid Sequence↗
PubMed1989

Primary structure of carboxypeptidase III from malted barley.

The primary structure of malt carboxypeptidase III has been determined. The enzyme is a single N-terminally blocked polypeptide chain containing 411 amino acid residues. The sequence of these amino acid residues was deduced from analysis of fragments of the polypeptide chain obtained by chemical cleavages with either cyanogen bromide or hydroxylamine and by enzymatic cleavages with either trypsin, S. aureus V8 protease or proteinase A from yeast. A glycosylated asparagine was found in position 71. The determined sequence was 97% homologous with the amino acid sequence derived from the nucleotide sequence of a gene coding for a wheat protein postulated to be a carboxypeptidase. The malt carboxypeptidase III sequence showed 34% homology with the amino acid sequence of the single-chain carboxypeptidase Y, and about 25% homology with the combined A- and B-chains of malt carboxypeptidase I and II as well as wheat carboxypeptidase II.

Amino Acid Sequence↗
PubMed1989

C-terminal sequence determination of peptides degraded with carboxypeptidases of different specificities and analyzed by 252-Cf plasma desorption mass spectrometry.

Identification of the truncated peptides by plasma desorption mass spectrometry in C-terminal sequence determination with carboxypeptidases offers several advantages over analysis of the liberated amino acids. It is possible to perform in situ digestion of a nitrocellulose-bound sample already used for molecular weight determination and thus obtain sequence information without further sample consumption. In time-course analysis the analytical information, although not obtained in real time, is sufficiently rapid to adjust the digestion conditions. There is no need for quantitation because the identification is based on molecular weight differences. Sensitivity in the low picomole range is obtainable. The digestion of a number of peptides (900-3500 Da) with carboxypeptidase Y and MII has been monitored. It was found that successive use of the enzymes or use of a mixture of the enzymes was often advantageous. The sequence of up to 10 residues from the C-terminus has been determined for the peptides studied.

Amino Acid Sequence↗
PubMed1988

Carboxypeptidase S-1 from Penicillium janthinellum: enzymatic properties in hydrolysis and aminolysis reactions.

Carboxypeptidase S-1 from Penicillium janthinellum has been isolated by affinity chromatography and characterized. The enzyme activity is unusually stable in organic solvents, e.g. 80% methanol. The hydrolysis of peptide substrates is apparently dependent on three ionizable groups. One group, with pKa of 4.0-4.5, is a catalytically essential residue in its deprotonated form, and another group with a pKa of 6.5-7.0 functions in its protonated form, apparently as the binding site for the C-terminal carboxylate group of peptide substrates. The third group, with a pKa of 5.0-5.5, appears to be a carboxylic acid group located at the S1 binding site. Deprotonation of this group to form a negatively charged carboxylate group has an adverse effect on the hydrolysis of substrates with hydrophobic amino acid residues at the P1 position and a beneficial effect on the hydrolysis of substrates with the positively charged arginyl or lysyl residues at this position. The substrate preference of the enzyme is consequently pH dependent. At pH 6.0 (the optimum for ester hydrolysis) the enzyme is essentially specific for Bz-X-OMe substrates where X = Arg and Lys. Using amino acids and amino acid amides as nucleophiles carboxypeptidase S-1 efficiently catalyses the formation of peptide bonds by aminolysis of peptides (transpeptidation reactions) and peptide esters provided that the substrate contains a basic amino acid residue at the P1 position, e.g. Bz-Arg-OBu and Bz-Arg-Leu-OH. With several nucleophiles the fractions of aminolysis exceed those previously reported in similar studies with carboxypeptidase Y and malt carboxypeptidase II.

Carboxypeptidases↗
PubMed1988

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

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 S1' 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 P1' position, i.e. -OBzl, -Val-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 leaving groups remained unaffected by such a group. The derivatives obtained by modification of the mutant enzyme with benzyl methanethiolsulfonate and methyl methanethiolsulfonate were effective in deamidations of peptide amides and peptide synthesis reactions, respectively.

Amides↗
PubMed1985

Tumor-derived angiogenesis factors from rat Walker 256 carcinoma: an experimental investigation and review.

Angiogenesis, the process of developing a hemovascular network, is an essential feature of the growth of solid tumors, and is induced by factors secreted by tumor cells. Assay procedures suitable for the investigation of angiogenesis, and for the screening of angiogenesis factors during purification are reviewed; and a number of reports describing the purification of angiogenesis factors, primarily from the rat Walker 256 carcinoma as starting material, are discussed. Work from the authors' laboratory is also presented. Walker 256 cells grown in large-scale culture were the source of a reproducible and homogeneous source of angiogenic material. Factors secreted by these cells were isolated by a series of chromatographic steps. Ion exchange chromatography on carboxymethyl-Sephadex produced two active fractions, one of which was fractionated into several macromolecular species by lectin affinity and hydrophobic adsorption chromatography. The other gave a high mol.wt, active fraction that was resolved into a low mol.wt, active component and a non-angiogenic but possibly carrier molecule with a mol.wt of 140,000. While none of the angiogenic factors were identified chemically, the results demonstrate the existence of both high and low mol.wt tumor-secreted angiogenic substances, confirming the hypothesis for tumor-induced angiogenesis and predicting potential means to interfere with the process of tumor growth.

Amnion↗
PubMedDate not supplied

Improvement of the applicability of carboxypeptidase Y in peptide synthesis by protein engineering.

Asn51 and Glu145 of (serine) carboxypeptidase Y function as binding sites for the C-terminal carboxylate group of peptide substrates, and Glu65 is involved in orienting these two amino acid residues. A series of mutants of carboxypeptidase Y where these three amino acid residues have been replaced were investigated for their applicability in transacylation reactions with amino acid esters as acceptors. With H-Val-OMethyl as the nucleophile, the fraction of aminolysis is significantly higher than with the corresponding amino acid, suggesting a beneficial effect of blocking the alpha-carboxylate group. Increasing the size of the alcohol moiety, i.e., -OEthyl, -OPropyl or OButyl, has an adverse effect on the binding of the nucleophile and on the maximum yield of aminolysis. Replacement of Asn51 and Glu145 with Ala or Gly has a pronounced beneficial effect both on binding and the maximum fraction of aminolysis. However, the results do not establish a specific type of interaction between the enzyme and these valine esters. It is probable that the rotational freedom around the ester bond allows multiple binding modes, depending on both the leaving group and type of structural change within the binding site. From a synthetic point of view, some of the mutant enzymes are much better than the wildtype enzyme when amino acid esters are used as nucleophiles.

Acylation↗