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At least 19 recordsLinked to original sources
PubMed1977

Studies on a carboxypeptidase Y mutant of yeast and evidence for a second carboxypeptidase Activity.

Immunological studies on the carboxypeptidase Y mutant prcl-l of Saccharomyces cerevisiae revealed the origin of mutation in the structural gene of carboxypeptidase Y. The absence of carboxypeptidase Y has no effect on growth, even after drastic changes of growth conditions. A double mutant (prc 1- leu2-) lacking carboxypeptidase Y and auxotrophic for leucine is able to grow on the peptide benzyloxycarbonylglycylleucine (Cbz-Gly-Leu) as sole nitrogen source, indicating the existence of a second carboxypeptidase. Using a new peptidase test, the existence of this second enzyme, called carboxypeptidase S, was confirmed biochemically.

Carboxypeptidases↗
PubMed1976

Carboxypeptidase inhibitor from potatoes. Interaction with derivatives of carboxypeptidase A.

The mechanism of action of a carboxypeptidase inhibitor from potatoes has been probed by studying its interaction with derivatives of carboxypeptidase A containing modified residues at the active site. Arsanilazocarboxypeptidase A, a derivative containing a chromophore attached to tyrosine 248, exhibits a circular dichroism spectrum which is sensitive to the presence of ligands at the active site (Kagan, H.M., and Vallee, B.L. (1969), Biochemistry 8, 4223). Since the spectral change attending binding of the carboxypeptidase inhibitor to arsanilazocarboxypeptidase A is similar to that produced by small substrates and inhibitors, the enzyme-inhibitor interaction also involves the enzyme active site. Catalytic activity is not required for inhibitor binding. Complexes of the inhibitor with apocarboxypeptidase A anc carboxypeptidase A which was inactivated by treatment with the affinity label, N-bromoacetyl-N-methyl-L-phenylalanine, are demonstrated by gel filtration experiments. Morever, competitive binding studies reveal that the latter derivative, in which the binding pocket is presumably blocked by reagent, binds inhibitor nearly as strongly as does the native enzyme, and differences in free energy of association being only 0.4 kcal/mol of a total binding energy of - 11 kcal/mol. A model is proposed to account for both the tight binding of inhibitor to the N-bromoacetyl-N-methyl-L-phenylalanine derivative and the involvement of the active site of arsanilazocarboxypeptidase A. It is suggested that the inhibitor fits into a shallow depression at the active site of the enzyme but does not penetrate into the binding pocket.

Binding Sites↗
PubMed1978

Stability of D-5,5-dimethyl-delta2-thiazoline-4-carboxylic acid in relation to its possible occurrence as a degradation product of penicillin by the exocellular DD-carboxypeptidase-transpeptidase from Streptomyces R61 and the membrane-bound dd-carboxypeptidase from Bacillus stearothermophilus.

The stability of D-5,5-dimethyl-delta2-thiazoline-4-carboxylic acid has been studied under various conditions. In 10 mM cacodylate, pH 6.5, and at 55 degrees C, D-5,5-dimethyl-delta2-thiazoline-4-carboxylic acid (at concentrations lower than 1 mM) is hydrolyzed into N-formyl-D-penicillamine with a half-life of 3 to 4 min. On this basis, it is very unlikely that D-5,5-dimethyl-delta2-thiazoline-4-carboxylic acid could be one of the end products resulting from the cleavage of benzylpenicillin by the DD-carboxypeptidase of Bacillus stearothermophilus (as reported by Hammarström and Strominger (1976) J. Biol. Chem. 251, 7947--7949). In 3 mM phosphate, pH 7.5, and at 37 degrees C, D-5,5-dimethyl-delta2-thiazoline-4-carboxylic acid (at concentrations lower than 1 mM) has a half-life of 45 min. On the basis of kinetic experiments carried out under these conditions with phenoxymethylpenicillin and the DD-carboxypeptidase-transpeptidase of Streptomyces R61, it is concluded that the primary product which arises from the thiazolidine moiety of the antibiotic molecule and gives rise to N-formyl-D-penicillamine, has a half-life of 10 min, a value which is not compatible with the hypothesis that D-5,5-dimethyl-delta2-thiazoline-4-carboxylic acid would be an intermediate involved in the fragmentation pathway.

Acyltransferases↗
PubMed1978

Enzymatically inactive, exchange-inert Co(III)-carboxypeptidase A: role of inner sphere coordination in peptide and ester catalysis.

Catalytically inactive, exchange-inert Co(III)-carboxypeptidase A has been prepared by reaction of Co(II)-carboxypeptidase A with the active-site-directed oxidizing agent m-chloroperbenzoic acid. Co(III)-carboxypeptidase A, isolated by affinity gel filtration chromatography, has the same amino acid composition and molecular weight as the starting material and contains 0.95 g-atom/mol of cobalt and 0.01 g-atom/mol of zinc. Its electron paramagnetic resonance, circular dichroic, magnetic circular dichroic, and visible absorption spectra are consistent with those of octahedral Co(III) model complexes. Co(III)-caboxypeptidase A is essentially devoid of catalytic activity toward both peptide and ester substrates of the native enzyme, and stopped-flow fluorescence studies with dansylated substrates show that it binds peptides, but not esters. Furthermore, the protein does not react with either type of substrate to yield a single turnover. The implications of these findings to the mechanism of action of carboxypeptidase A are discussed in the light of the "metal-carbonyl" and "metal-hydroxide" hypotheses. Since Co(III)-carboxypeptidase A does not bind esters, inner-sphere coordination to the metal appears to be necessary for ester binding. All attempts to prepare Co(III)-carboxypeptidase A by treatment of Co(II)-carboxypeptidase A with hydrogen peroxide according to previously published procedures (Kang, E.P., Storm, C.B., & Carson, F.W. (1975) J. Am. Chem. Soc. 97, 6723) have been unsuccessful, and the present results do not confirm earlier reports that Co(III)-carboxypeptidase A exhibits esterase activity or that its activity is dependent on the method of preparation of the precursor Co(II)-carboxypeptidase A (Jones, M.M., Hunt, J.B., Storm, C.B., Evans, P.S., Carson, F.W. & Pauli, W.J. (1977) Biochem. Biophys. Res. Commun. 75, 253). These findings call for a reexamination of mechanistic conclusions based on the assumption that Co(III)-carboxypeptidase A is an active esterase.

Apoenzymes↗
PubMed1976

Human carboxypeptidase B. II. Purification of the enzyme from pancreatic tissue and comparison with the enzymes present in pancreatic secretion.

Carboxypeptidase B (peptidyl-L-lysine (-L-arginine) hydrolase, EC 3.4.12.3) has been isolated and purified to apparent homogeneity from activated extracts of human pancreas tissue. The purified enzyme has been shown to be a single polypeptide of 34 000 daltons. In this respect the enzyme from pancreatic tissue, designated native human carboxypeptidase B, differs from the two forms present in human pancreatic juice (fractions I and II), both of which are composed of two polypeptides of approximately 24 000 and 9000 daltons. In addition, the three forms of human carboxypeptidase B differ in electrophoretic mobility in polyacrylamide gel electrophoresis and in chromatographic behavior on DEAE-cellulose. Two immunological methods, micro-complement fixation and radioimmunoassay, have shown a high degree of structural similarity between the three forms of human carboxypeptidase B. Micro-complement fixation experiments indicate that the amino acid sequences of the three enzymes differ by less than one percent. In vitro digestion studies have indicated that trypsin alone is sufficient to convert native carboxypeptidase B to carboxypeptidase B II. However, no combination of trypsin, chymotrypsin, and/or elastase was capable of converting native carboxypeptidase B to carboxypeptidase B I in vitro.

Amino Acids↗
PubMed1978

Studies on the carboxypeptidase Y-inhibitor complex of yeast.

We report in vitro studies on the interaction of several substrates with the carboxypeptidase Y-inhibitor complex of yeast. Inhibition of carboxypeptidase Y cleavage of two peptides by carboxypeptidase Y-inhibitor is shown to be competitive. The experiments show a wide variation in the degree of cleavage of a variety of peptide substrates by carboxypeptidase Y, despite the presence of the inhibitor protein. The most likely explanation for this behaviour is a different capacity for the peptides to dissociate the inhibitor protein from the substrate-binding site of carboxypeptidase Y. While the carboxypeptidase Y-inhibitor is insensitive to proteolytic inactivation when complexed with carboxypeptidase Y, it is sensitive when in the free state. Addition of the substrate, N-Cbz-Phe-Leu, to the carboxypeptidase Y-inhibitor complex, however, allows proteolytic inactivation of the inhibitor protein. We suggest that the proteinase-inhibitor may play a crucial role in the regulation of proteinase activity. The inhibitor protein generally protects proteins from unwanted proteinase action. However, it will allow cleavage of proteins which, by some signal triggered metabolically, become substrates due to the exposure of amino acid sequences normally buried, and exhibiting a high affinity for the proteinase.

Carboxypeptidases↗
PubMed1977

Purification of carboxypeptidase B from human pancreas.

Carboxypeptidase B of the human pancreas was purified by chromatography on DEAE-cellulose and CM-cellulose columns. Two forms of the enzyme, named carboxypeptidase B1 and B2, were separated. They have similar mol.wts. (34250 +/- 590) as established by polyacrylamide-gel disc electrophoresis and by gel filtration. Carboxypeptidase B2 migrates further towards the anode in disc electrophoresis. When the amino acid content of the enzymes was analysed, carboxypeptidase B2 had four more glycine and three more aspartic acid residues than had form B1. The amino acid sequence of the human carboxypeptidase B1 differs from that of the bovine enzyme only in two places in the N-terminal 20-amino-acid sequence. The N-terminal amino acid in carboxypeptidase B1 and B2 is alanine. The peptide 'map' of the tryptic digest of carboxypeptidase B1 contained more peptides than did that of form B2. The Km, the Vmax. and the pH optimum of the cleavage of the peptide substrate hippurylarginine and the ester substrate hippurylargininic acid were similar for both enzymes. CoCl2 accelerated the peptidase activity, and cadmium acetate enhanced the esterase activity, of human carboxypeptidases B1 and B2. Urea and sodium dodecyl sulphate inhibited the enzymes.

Amino Acid Sequence↗
PubMed1977

Basis for the observed fluctuation of carboxypeptidase II activity during the cell cycle in BUG 6, a temperature-sensitive division mutant of Escherichia coli.

Diaminopimelyl-d-alanyl carboxypeptidase (carboxypeptidase II) is most active at the time of division, whether measured in toluene-treated cells of Escherichia coli K-12 strain D11-1, fractionated by size, or in toluene-treated cells of the temperature-sensitive division mutant, BUG 6 (B. D. Beck and J. T. Park, 1976). The present investigation has now shown that, under conditions that permit division, the increased carboxypeptidase II activity in toluenetreated cells of BUG 6 is probably not due to protein synthesis. Although dividing cells are more permeable than nondividing cells, permeability differences are not sufficient to account for the changes in carboxypeptidase II activity. Thus, in the toluene-treated nondividing cells, carboxypeptidase II is present, but its activity is masked, which suggests the presence of an inhibitor. Another striking difference between nondividing and dividing cells is that carboxypeptidase II is much more readily released from dividing cells by both tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetic acid and toluene treatment. Carboxypeptidase II was partially purified and found to be an 86,000-molecular-weight protein consisting of two 43,000-molecular-weight polypeptides. Tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetic acid treatment of nondividing cells releases less than 10% of the carboxypeptidase II and other periplasmic proteins that are releasable from dividing cells.

Carboxypeptidases↗
PubMed1975

Evidence for an essential histidine in carboxypeptidase Y. Reaction with the chloromethyl ketone derivative of benzyloxycarbonyl-L-phenylalanine.

The possible role of histidine residues in the catalytic function of carboxypeptidase Y from bakers' yeast has been investigated using site-specific reagents. Among the reagents tested, benzyloxy-L-phenylalanylchloromethane (Z-PheCH2Cl) was the most powerful inhibitor of the enzyme. It irreversibly inactivated both the peptidase and esterase activities with an apparent second order rate constant of 3.8 M-minus 1 S-minus 1; the D isomer caused essentially no effect on either activity. Inhibition by L-Z-PheCH2Cl, the reaction retarded by certain competitive inhibitors of the enzyme. Using radioactive L-Z-PheCH2Cl, the reaction with the enzyme was shown to be essentially stoichiometric. Diisopropylphosphorofluoridate (iPr2PF)-inactivated enzyme failed to react with Z-PheCH2Cl, and conversely, the Z-PheCH2Cl-inhibited enzyme failed to react with radioactive iPr2PF. Amino acid analyses of the Z-PheCH2Cl-inactivated enzyme revealed the loss of essentially 1 residue, with a concomitant yield of a 0.62 residue of N-t-carboxymethylhistidine. Since carboxypeptidase Y has a reactive serine at its active center, we concluded from these results that the mechanism involves a charge-relay system in the hydrolysis of peptide and ester substrates, as in chymotrypsin. An -SH group of carboxypeptidase Y was not affected during the reaction with L-Z-PheCH2Cl. The generic name "serine carboxypeptidase" has been proposed for carboxypeptidase Y and for the iPr2PF-sensitive carboxypeptidases from plants, molds, and animal tissues, in order to distinguish them from "metal carboxypeptidase" to which carboxypeptidase A (EC 3.4.12.2) and B (EC 3.4.12.3) belong.

Amino Acids↗
PubMed1978

Isolation of a mutant of Escherichia coli lacking penicillin-sensitive D-alanine carboxypeptidase IA.

A mutant of Escherichia coli that is deficient in D-alanine carboxypeptidase IA has been isolated. The enzyme is membrane bound and moderately sensitive to penicillin. It catalyzes in vitro both D-alanine carboxypeptidase and transpeptidase reactions. Being able to synthesize crosslinked peptidoglycan both in vivo and in vitro despite the absence of enzyme activity, the newly isolated mutant grew normally under a wide range of growth conditions. Therefore, this enzyme, like D-alanine carboxypeptidase IB, is not required for normal peptidoglycan synthesis in E. coli. The defect in the activity of D-alanine carboxypeptidase IA in the mutant however was not associated with disappearance of penicillin-binding proteins 5 and 6 (which have been shown to be D-alanine carboxypeptidase IA) or any of the other protein bands that bind [14C]penicillin G. Genetic mapping studies showed that the mutation (dacA) is located close to leuS(13.7 min) on the E. coli chromosome map. Double mutants (dacA dacB) that are deficient in both D-alanine carboxypeptidases IA and IB were obtained. These double mutants also were found to grow normally and to catalyze normal formation of crosslinked peptidoglycan.

Carboxypeptidases↗
PubMed1975

Amino-acid sequence of bovine carboxypeptidase B.

The amino-acid sequence of bovine carboxypeptidase B [peptidyl-L-lysine(-L-arginine)hydrolase, EC 3.4.12.3] has been determined using the heavy and light chains of the enzyme isolated from spontaneously activated pancreatic juice. Comparison of the sequence with that of carboxypeptidase A shows that the two enzymes are homologous (49% identity) and that all but one of the functional residues identified in carboxypeptidase A occur in corresponding loci in carboxypeptidase B (peptidyl-L-amino acid hydrolase, EC 3.4.12.2). The exception is the replacement of Ile-255 at the bottom of the substrate binding pocket of carboxypeptidase A, by aspartic acid in carboxypeptidase B. This single change can account for the difference in specificity of the two enzymes.

Amino Acid Sequence↗