Serine Carboxypeptidase Research Articles

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)

Structure of wheat serine carboxypeptidase II at 3.5-A resolution. A new class of serine proteinase.

Structure of wheat serine carboxypeptidase II at 3.5-A resolution. A new class of serine proteinase.

Mouse "protective protein". cDNA cloning, sequence comparison, and expression.

The "protective protein" is the glycoprotein that forms a complex with the lysosomal enzymes beta-galactosidase and neuraminidase. Its deficiency in man leads to the metabolic storage disorder galactosialidosis. The primary structure of human protective protein, deduced from its cloned cDNA, shows homology to yeast serine carboxypeptidases. We have isolated a full-length cDNA encoding murine protective protein. The nucleotide sequences as well as the predicted amino acid sequences are highly conserved between man and mouse. Domains important for the protease function are completely identical in the two proteins. Both human and mouse mature protective proteins covalently bind radiolabeled diisopropyl fluorophosphate. Transient expression of the murine cDNA in COS-1 cells yields a protective protein precursor of 54 kDa, a size characteristic of the glycosylated form. This cDNA-encoded precursor, endocytosed by human galactosialidosis fibroblasts, is processed into a 32- and a 20-kDa heterodimer and corrects beta-galactosidase and neuraminidase activities. A tissue-specific expression of protective protein mRNA is observed when total RNA from different mouse organs is analyzed on Northern blots.

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 12 12 or enantiomorph with cell dimensions of a = b = 98.2 A ̊ and c = 209.5 A ̊ . The crystals diffract to about 2.3 Å resolution using rotating-anode X-ray generators. Assuming a dimer of M r 120,000 in the asymmetric unit, V m = 2.1 A ̊ 3/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 °C, pH 6.4 to 8.0.

Carboxypeptidase Y-Catalyzed Reaction in Systems Containing Organic Solvent

The effect of organic solvents on carboxypeptidase Y (a serine carboxypeptidase from yeast)-catalyzed hydrolysis of amino acid ester and peptide synthesis from N-acyl amino acid ester and amino acid amide was investigated.The Km value of ester hydrolysis increased with an increase in the solvent content. Dioxane was the most effective and dimethyl sulfoxide (DMSO) the least, whilst Kcat showed a tendency to increase slightly in N, N-dimethylformamide (DMF) and DMSO. For dioxane and acetonitrile (MeCN) a maximum was observed.In peptide formation from Fua-Phe-OEt and Gly-NH2, dioxane and MeCN supported high product yield at molar fractions smaller than ca. 0.05 but the yield decreased significantly at higher fractions, although a relatively constant selectivity (ratio of the peptide bond formed to the ester consumed) was maintained. DMSO gave rather low peptide yields and selectivity even at lower molar fractions. DMF showed an intermediate tendency.An apparent saturation parameter of the amine component wa...

生酒に含まれる各種酵素および微生物の加圧失活

Hydrostatic pressure of 1000_??_6000kg/cm2 was applied to enzyme solution and Namazake (nonpasteurized rice wine) to investigate high pressure effects on inactivation of enzymes and microorganisms. The pressurization caused a partial inactivation of enzymes (α-amylase, glucoamylase, protease, serine carboxypeptidase), and after pressurization at 6000kg/cm2 for 10min at 25°C, 20_??_60 percent of the original activity remained. Increasing temperature, ethanol concentration, and duration of pressurization accelerated the inactivation of these enzymes. Repeating the pressurization was also effective in increasing enzyme inactivation. Pressurization of 3000kg/cm2 for 10min at 25°C sterilized the Lactobacillus and yeast in Namazake. Namazake after pressurization at 4000kg/cm2 for 30min at 25°C had the same flavor as the original Namazake. This was stored for a month at 30°C and no deterioration in constituents or flavor was noted.

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.

Characterization of the yeast KEX1 gene product: a carboxypeptidase involved in processing secreted precursor proteins.

We have identified and partially characterized the Saccharomyces cerevisiae KEX1 gene product, Kex1p, to assess its role in processing secreted protein precursors. Anti-Kex1p antibodies identified a 113-kilodalton protein that was absent in cells in which the KEX1 gene has been disrupted and that was more abundant in cells overexpressing the KEX1 gene. Kex1p was found to be a membrane-associated glycoprotein with N-linked carbohydrate. The N-linked oligosaccharide(s) was modified in a progressive manner after synthesis, causing the glycoprotein to slowly increase in mass to 115 kilodaltons. After a Kex2p-mediated cleavage event at specific pairs of basic amino acids, alpha-factor and K1 killer toxin precursors have COOH-terminal dibasic residue extensions and require a carboxypeptidase B-like enzyme to process the precursors to maturity. A carboxypeptidase activity, with apparent specificity for basic amino acids, was detected in KEX1 cells. Disruption of the KEX1 gene abolished this activity, while overexpression of KEX1 increased it. Our results provide biochemical evidence consistent with earlier genetic work, that KEX1 encodes a serine carboxypeptidase involved in the processing of precursors to secreted mature proteins.

Characterization of the yeast KEX1 gene product: a carboxypeptidase involved in processing secreted precursor proteins.

We have identified and partially characterized the Saccharomyces cerevisiae KEX1 gene product, Kex1p, to assess its role in processing secreted protein precursors. Anti-Kex1p antibodies identified a 113-kilodalton protein that was absent in cells in which the KEX1 gene has been disrupted and that was more abundant in cells overexpressing the KEX1 gene. Kex1p was found to be a membrane-associated glycoprotein with N-linked carbohydrate. The N-linked oligosaccharide(s) was modified in a progressive manner after synthesis, causing the glycoprotein to slowly increase in mass to 115 kilodaltons. After a Kex2p-mediated cleavage event at specific pairs of basic amino acids, alpha-factor and K1 killer toxin precursors have COOH-terminal dibasic residue extensions and require a carboxypeptidase B-like enzyme to process the precursors to maturity. A carboxypeptidase activity, with apparent specificity for basic amino acids, was detected in KEX1 cells. Disruption of the KEX1 gene abolished this activity, while overexpression of KEX1 increased it. Our results provide biochemical evidence consistent with earlier genetic work, that KEX1 encodes a serine carboxypeptidase involved in the processing of precursors to secreted mature proteins.

Identification of carboxypeptidase and tryptic esterase activities that are complexed to proteoglycans in the secretory granules of human cloned natural killer cells.

Human cloned 35S-labeled NK cells were disrupted by nitrogen cavitation, and their secretory granules were obtained by filtration through 5-micron and 3-micron membrane filters followed by Percoll density-gradient centrifugation. These granule preparations, which contained 35S-labeled chondroitin sulfate A proteoglycans, were sonicated and were analyzed for carboxypeptidase activity and tryptic serine esterase activity. A carboxypeptidase activity that digested angiotensin I to des-Leu-angiotensin I, Ile-His-Pro-Phe to Ile-His-Pro and Phe, and hippuryl-L-phenylalanine to hippuric acid and Phe was detected in the granules of these NK cells. As determined by cleavage of the tetrapeptide, the pH optimum of the carboxypeptidase was 7.0. As assessed by the cleavage of N-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLTe), the granule preparations also contained a serine esterase with trypsin-like specificity that had a pH optimum of 8.5. When the isolated secretory granules were disrupted and chromatographed on columns of Sepharose CL-2B in PBS, greater than 60% of the BLTe serine esterase activity and essentially all of the carboxypeptidase activity filtered as a macromolecular complex with approximately 8% of the 35S-labeled proteoglycans. Whereas treatment with 4 M urea or nonionic detergent failed to disrupt the macromolecular complex, the serine esterase activity was dissociated from the macromolecular complex in the presence of 3 M NaCl, demonstrating an ionic interaction with the proteoglycans. No difference was observed in the disaccharide composition of the chondroitin sulfate glycosaminoglycans of the 35S-labeled proteoglycans that were complexed with the enzymes as compared to those that were not complexed. These studies indicate that the secretory granules of human NK cells contain serine esterase activity and carboxypeptidase activity, both of which have neutral pH optima, and both of which are bound to protease-resistant chondroitin sulfate proteoglycans.

Wheat Seed Carboxypeptidase and Joint Action on Gliadin of Proteases from Dry and Germinating Seeds

A carboxypeptidase preparation, homogeneous according to polyacrylamide gel electrophoresis and ultracentrifugation, was obtained from wheat seeds. The isolation procedure included (NH4)2SO4 fractionation, gel-filtration on Sepharose-6B and affinity chromatography on CABS-Sepharose. Mr of the enzyme determined by gel-filtration was 126 000. The enzyme consisted of two non-identical subunits of Mr 60 000 and 63 000. The pl of the carboxypeptidase was 5.7. The inhibitory analysis revealed that the isolated enzyme is a serine carboxypeptidase. The carboxypeptidase preferentially hydrolysed N-substituted dipeptides with aromatic amino acid residues at the C-terminus and showed weak hydrolysing activity towards gliadin. The combined action of carboxypeptidase and aspartic proteinase from dry wheat seeds led to an increase in the degree of proteolysis of the storage protein compared to that resulting from the sum of the action of individual enzymes. The cysteine proteinase from germinated wheat seeds caused complete degradation both of gliadin, which had first been treated with proteases of dry seeds (aspartic proteinase plus carboxypeptidase), and of untreated gliadin. However, when gliadin had first been hydrolysed with dry seed proteases, the rate of its proteolysis with cysteine proteinase increased 3–4 times compared to the non-hydrolysed gliadin. The data indicate the importance of preliminary modification of gliadin with dry wheat proteases, which, apparently, enhances the supply of nutritive substances to the embryo in the course of seed germination.

Action of serine carboxypeptidase from Aspergillus saitoi on carboxyterminal amidated peptides.

The specificity of the serine carboxypeptidase from Aspergillus saitoi (EC 3.4.16.1) was investigated on carboxyterminal amidated peptides such as benzyloxycarbonyl(Z)-Ala-Phe-NH2, gastrin-related peptide, molluscan cardioexcitatory neuropeptide, eledoisin-related peptide, and (D-Ala2, Met5)-enkephalinamide. The enzyme did not hydrolyze Z-Ala-Phe-NH2. It acted only as a carboxyamidase for the carboxyterminal peptide bond of gastrin-related peptide and as an amidase for enkephalinamide. The enzyme had both carboxyamidase activity and amidase activity for molluscan cardioexcitatory neuropeptide and eledoisin-related peptide. Whether the enzyme acts as an amidase or carboxyamidase, the hydrophobicity of P3 and P4 positions of the substrate may be important. After removal of the carboxyterminal amino acid amide and/or ammonia, the enzyme catalyzed the sequential liberation of the amino acid from the carboxyterminus of the substrate.

361 Cloned human natural killer cells contain serine proteases, carboxypeptidases, and proteoglycans in their secretory granules

361 Cloned human natural killer cells contain serine proteases, carboxypeptidases, and proteoglycans in their secretory granules

Isolation of carboxypeptidase III from malted barley by affinity chromatography

A novel serine carboxypeptidase has been isolated from malted barley by affinity chromatography. The enzyme has been termed malt carboxypeptidase III in correspondence with the nomenclature suggested byMikola 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 pKa 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 1 ′ position and Phe, Leu, Met and Ala in the P1 position. The enzyme is inhibited by diisopropyl phosphorofluoridate, mercuric ions and various organomercuric compounds.

Action of Serine Carboxypeptidases on Endopeptidase Substrates, N-Acyldipeptideamides

Abstract Action patterns of two serine carboxypeptidases, one from yeast (Y) and the other from wheat bran (W), on N-[3-(2-furyl)acryloyl]-(Fua-) dipeptideamide substrates were examined by HPLC and amino acid analysis. In the reaction of the wheat enzyme the substrates were hydrolyzed to Fua-amino acid and no sufficient amount of Fua-dipeptides were detected on HPLC in the product mixtures. Very few or no free amino acids were observed by amino acid analysis. This indicates that the wheat enzyme exhibited carboxamidopeptidase activity on these substrates. On the contrary, carboxypeptidase Y gave Fua-amino acids and Fua-dipeptides as products, depending on the structure of the substrates. Accordingly, liberations of free amino acids were detected in some cases. This result shows that the yeast enzyme acts on some of the substrates in a two step manner: First by amidase and second by a carboxypeptidase activity. Based on these results the substrate binding mechanisms of these enzymes are discussed.

Determination of C-terminal sequences by digestion with serine carboxypeptidases: The influence of enzyme specificity

It is demonstrated that C-terminal sequence determinations of a number of proteins and peptides by digestion with serine carboxypeptidases often require use of different reaction conditions and/or use of carboxypeptidases with different specificities. The commonly utilized carboxypeptidase Y from yeast with preference for hydrophobic amino acid residues is conveniently supplemented by carboxypeptidase II from amlt which includes Lys and Arg in its specificity range. This renders it particularly suitable for C-terminal digestion of tryptic peptides. In some cases a phenylmercuric chloride modified derivative of carboxypeptidase Y which has a decreased preference for hydrophobic amino acid residues can be used with advantage.

Degradation of storage proteins in germinating seeds

The criteria for the participation of proteases in the mobilization of storage proteins during seed germination are formulated. The proteases that satisfy these criteria, namely the acid cysteine endopeptidases, serine carboxypeptidases and neutral aminopeptidases, are reviewed. The possibility of other seed proteases participating in storage protein degradation is discussed. The course of 11S and 7S storage protein degradation through the action of endogenous and exogenous proteases is described. The 11S and 7S proteins are modified during the early stages of proteolysis and the effects of these modifications on the regulation of breakdown are examined. A scheme for 11S protein degradation in germinating seeds is presented.

Synthesis and antitumor activity of spergualin analogues. III. Novel method for synthesis of optically active 15-deoxyspergualin and 15-deoxy-11-O-methylspergualin.

Optically active 15-deoxyspergualin (II) and 15-deoxy-11-O-methylspergualin (IIa) were synthesized, and their antitumor activities were examined. The (-)-enantiomers of both II and IIa were active against mouse leukemia L1210, while the (+)-enantiomers were almost inactive. The optical resolution of the key intermediate, (+/-)-N-(7-guanidinoheptanoyl)-alpha-alkoxyglycine (VI) was achieved by use of an exopeptidase, serine (acid) carboxypeptidase [EC 3.4.16.1] and (+/-)-N-(7-guanidinoheptanoyl)-alpha-alkoxyglycyl-L-amino acid (VIII) as the substrate. Considering the enzymatic susceptibility of the substrate (VIII), we deduced that the absolute configuration of the carbon at 11 (C-11) of the bioactive (-)-enantiomer, and so that of natural spergualin (I), is S. This is, to our knowledge, the first report of the use of carboxypeptidase for the resolution of N-acyl amino acid.

Endopeptidase and Carboxypeptidase Enzymes of Vacuoles Prepared from Mesophyll Protoplasts of the Primary Leaf of Wheat Seedlings

Summary Vacuoles prepared from mesophyll protoplasts of the primary leaf of wheat ( Triticum aestivum L. cv. Egret) contain carboxypeptidase (CP) and endopeptidase (EP) activity. CP activity was optimal at pH 5.2. The most preferred substrate was Z-Phe-Ala and activity against a wide range of other substrates was dependent on the C-terminal amino acid. Z-dipeptides with proline in any position were not hydrolysed. CP activity was inhibited by phenylmethylsulfonyl fluoride, diisopropylfluorophosphate, sodium dodecyl sulfate (SDS) and 4-hydroxymercuric benzoic acid. Thus the CP enzyme from the wheat leaf is a Serine Carboxypeptidase (EC 3.4.16). EP activity, assessed by following the degradation of the Large Sub Unit (M r 53.1 kD) of Ribulose 1,5-Bisphosphate Carboxylase (RuBPCase) by SDS-PAGE, was optimal at pH 5.2 (-SDS) and 5.5 (+SDS). The overall rate of degradation of the Large Sub Unit (LSU) was increased about 2.6-fold by the addition of 0.2 % SDS to the assay medium. In the absence of SDS there were only three prominent polypeptide breakdown products, M r 52.1, 50.9 and 50.9 kD. The 50.9 and 50.1 kD products appeared resistant to further hydrolysis, whilst the 52.1 kD product was eventually degraded. The presence of SDS increased the heterogeneity of the breakdown products, polypeptides with M r 51.8, 44.4, 43.6, 30.3 and 29.5 kD were especially prominent. The major portion of the vacuolar EP activity was considered to belong to the Thiol proteinase group (EC 3.4.22), although indirect evidence was obtained for two other EP activities. There appeared to be little co-operation between the EP and CP activities in the degradation of RuBPCase.

Serine carboxypeptidases. A review

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 pKa 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 of peptide esters can be employed to exchange C-terminal amino acid residues in peptides and in step-wise synthesis of polypeptides, respectively. The type of reactions catalyzed by these enzymes is limited by their specificities but, fortunately, some of the derivatives of carboxypeptidase Y with changed specificity due to chemical modifications and genetic substitutions of amino acid side-chains located at binding sites can be employed with advantage. These modified enzymes are examples on how the different activities of an enzyme can be perturbed by “protein engineering”, hence rendering the enzyme particularly suitable for certain processes.