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PubMed1988

Carboxypeptidase H-like immunoreactivity in the striatum of cats and monkeys.

The enzyme carboxypeptidase H was detected by immunohistochemistry in the striatum of adult cats and monkeys. Specific labelling was observed in the neuropil as well as in both medium-sized and large neuronal cell bodies. The distribution of neurons and neuropil expressing immunoreactivity to carboxypeptidase H was examined in relation to the pattern of immunoreactivity to the neuropeptides enkephalin and substance P. Carboxypeptidase H-like immunoreactivity was found both in zones rich and poor in immunostaining for the two peptides, but was usually denser in those striatal areas in which substance P-positive cell bodies are clustered (striosomes). The results further suggest a role for carboxypeptidase H in the metabolism of multiple neuropeptides in vivo.

Animals↗

PubMed1990

Distribution of carboxypeptidase H messenger RNA in rat brain using in situ hybridization histochemistry: implications for neuropeptide biosynthesis.

Carboxypeptidase H is an exopeptidase which is highly specific for C-terminal basic amino acids and thought to play a role in neuropeptide biosynthesis. The distribution of carboxypeptidase H mRNA was examined in adult rat brain using in situ hybridization histochemistry. Enzyme transcripts were detected in all major brain areas. Very high levels of carboxypeptidase H mRNA were found in the hippocampus associated with pyramidal cells. Other brain regions showed varied levels of labelling, ranging from high levels in the magnocellular cells of hypothalamic nuclei to very low levels in the striatum. The appearance of enzyme transcripts throughout brain supports a role for this enzyme in the biosynthesis of many neuropeptides. The expression of transcripts in certain ventricular ependymal cells identified them as a new potential peptidergic cell type. The variations in levels of expression of carboxypeptidase H mRNA may reflect differences in peptidergic activity in different neuronal systems.

Animals↗

PubMed1996

Proinsulin cleaved by furin is processed to chromatographically mature insulin by carboxypeptidases in nonneuroendocrine cells.

Proinsulin is converted to mature insulin by two reactions, cleavage by the prohormone convertases PC2 and PC3, and removal of basic residues by carboxypeptidase H. These reactions are performed in the secretory granules of pancreatic beta cells. When we replaced the processing sites of proinsulin with furin-cleavable sites, the three nonneuroendocrine cell lines Hep G2, CHO, and NIH/3T3 produced insulin with the same size as synthetic human insulin. Although the three cell lines expressed different quantities of carboxypeptidase H mRNA, the cytosol fractions of the cells exhibited similar levels of carboxypeptidase activity, suggesting that additional carboxypeptidases were active. The insulins resulting from the three cell lines were eluted as a single peak on a cation-exchange chromatography column, indicating that proinsulin can be maturated to insulin even in nonneuroendocrine cells.

3T3 Cells↗

PubMed1985

Alterations of serum carboxypeptidases N and angiotensin-I-converting enzyme in malignant diseases.

The activities of serum carboxypeptidases N (E.C. 3.4.17.3) and serum angiotensin-I-converting enzyme (E.C. 3.4.15.1) were spectrophotometrically measured in patients with hematological disorders and in histologically proven bronchogenic carcinomas. The enzyme activities have been compared with various laboratory parameters. Controls were healthy subjects. No significant sex differences of carboxypeptidases N and angiotensin-I-converting enzyme were found in the healthy controls or in the whole patients' group. The carboxypeptidases N were significantly elevated in patients with lung cancer (p less than 0.0001) compared to the healthy controls. The angiotensin-I-converting enzyme was significantly reduced (p less than 0.0001) in serum of patients with bronchogenic carcinoma. In responders to chemo- and/or radiotherapy, the carboxypeptidases decreased and the angiotensin-I-converting enzyme increased to normal enzyme values. In non-responders to these therapies no change in these enzymes could be observed.

Adenocarcinoma↗

PubMed2003

Carboxypeptidase B inhibitors reduce tissue factor-induced renal microthrombi in rats.

Procarboxypeptidase B (also known as thrombin-activatable fibrinolysis inhibitor) is a recently described plasma zymogen known to be activated by thrombin in plasma. Carboxy-terminal lysine residues from partially degraded fibrin are important for the binding and activation of plasminogen, and carboxypeptidase B, an active form of procarboxypeptidase B, has been shown to inhibit fibrinolysis by eliminating these residues. The present paper investigates the effects of carboxypeptidase B inhibitors, DL-mercaptomethyl-3-guanidinoethylthiopropanoic acid (MGPA) and potato-derived carboxypeptidase inhibitor (CPI), on tissue factor (TF)-induced microthrombosis in rats. Intravenous injection of MGPA (3 mg/kg and higher) or CPI (0.3 mg/kg and higher) after microthrombi formation dramatically attenuated TF-induced glomerular fibrin deposition with an increase in plasma levels of D-dimer. These results indicate that carboxypeptidase B inhibitors can enhance endogenous fibrinolysis and reduce thrombi in the TF-induced microthrombosis model after systemic administration even after thrombi formation.

3-Mercaptopropionic Acid↗

PubMed1983

Refined crystal structure of carboxypeptidase A at 1.54 A resolution.

The crystal structure of bovine carboxypeptidase A (Cox) has been refined at 1.54 A resolution using the restrained least-squares algorithm of Hendrickson & Konnert (1981). The crystallographic R factor (formula; see text) for structure factors calculated from the final model is 0.190. Bond lengths and bond angles in the carboxypeptidase A model have root-mean-square deviations from ideal values of 0.025 A and 3.6 degrees, respectively. Four examples of a reverse turn like structure (the "Asx" turn) requiring an aspartic acid or asparagine residue are observed in this structure. The Asx turn has the same number of atoms as a reverse turn, but only one peptide bond, and the hydrogen bond that closes the turn is between the Asx side-chain CO group and a main-chain NH group. The distributions of CO-N and NH-O hydrogen bond angles in the alpha-helices and beta-sheet structures of carboxypeptidase A are centered about 156 degrees. A total of 192 water molecules per molecule of enzyme are included in the final model. Unlike the hydrogen bonding geometry observed in the secondary structure of the enzyme, the CO-O(wat) hydrogen bond angle is distributed about 131 degrees, indicating the role of the lone pair electrons of the carbonyl oxygen in the hydrogen bond interaction. Twenty four solvent molecules are observed buried within the protein. Several of these waters are organized into hydrogen-bonded chains containing up to five waters. The average temperature factor for atoms in carboxypeptidase A is 8 A2, and varies from 5 A2 in the center of the protein, to over 30 A2 at the surface.

Amino Acid Sequence↗

PubMed1985

alpha-Bromo ketone substrate analogues are powerful reversible inhibitors of carboxypeptidase A.

The aldehyde (RS)-2-benzyl-4-oxobutanoic acid, which is 25% hydrated at pH 7.5, has recently been shown to be a strong reversible competitive inhibitor of carboxypeptidase A [Ki = 0.48 nM; Galardy, R. E., & Kortylewicz, Z. P. (1984) Biochemistry 23, 2083-2087]. The ketone analogue of this aldehyde (RS)-2-benzyl-4-oxopentanoic acid (IV) is not detectably hydrated under the same conditions and is 1500-fold less potent (Ki = 730 microM). The ketone homologue (RS)-2-benzyl-5-oxohexanoic acid (XIII) is also a weak inhibitor (Ki = 1.3 mM). The alpha-monobrominated derivatives of these two ketones are, however, strong competitive inhibitors with Ki's of 0.57 microM and 1.3 microM, respectively. Oximes derived from the aldehyde, the ketones IV and XIII, and a homologue of the aldehyde are weak inhibitors with Ki's ranging from 480 to 7900 microM. The inhibition of carboxypeptidase A by the alpha-monobrominated ketones is reversible and independent of the time (up to 6 h) of incubation of enzyme and inhibitor together. Bromoacetone at a concentration of 30 mM does not inhibit carboxypeptidase A. Incubation of an equimolar mixture of 2-benzyl-4-bromo-5-oxohexanoic acid (XV) and enzyme for 1 h led to the recovery of 82% of XV, demonstrating that it is the major species reversibly bound during assay of inhibition. Taken together, these results indicate that tight binding of carbonyl inhibitors to carboxypeptidase A requires specific binding of inhibitor functional groups such as benzyl and an electrophilic carbonyl carbon such as that of an alpha-bromo ketone or aliphatic aldehyde.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Crystal structure of the complex of carboxypeptidase A with a strongly bound phosphonate in a new crystalline form: comparison with structures of other complexes.

O-[[(1R)-[[N-(Phenylmethoxycarbonyl)-L-alanyl]amino]ethyl] hydroxyphosphinyl]-L-3-phenyllacetate [ZAAP(O)F], an analogue of (benzyloxycarbonyl)-Ala-Ala-Phe or (benzyloxycarbonyl)-Ala-Ala-phenyllactate, binds to carboxypeptidase A with great affinity (Ki = 3 pM). Similar phosphonates have been shown to be transition-state analogues of the CPA-catalyzed hydrolysis [Hanson, J. E., Kaplan, A. P., & Bartlett, P. A. (1989) Biochemistry 28, 6294-6305]. In the present study, the structure of the complex of this phosphonate with carboxypeptidase A has been determined by X-ray crystallography to a resolution of 2.0 A. The complex crystallizes in the space group P2(1)2(1)2(1) with cell dimensions a = 61.9 A, b = 67.2 A, and c = 76.2 A. The structure of the complex was solved by molecular replacement. Refinement of the structure against 20,776 unique reflections between 10.0 and 2.0 A yields a crystallographic residual of 0.193, including 140 water molecules. The two phosphinyl oxygens of the inhibitor bind to the active-site zinc at 2.2 A on the electrophilic (Arg-127) side and 3.1 A on the nucleophilic (Glu-270) side. Various features of the binding mode of this phosphonate inhibitor are consistent with the hypothesis that carboxypeptidase A catalyzed hydrolysis proceeds through a general-base mechanism in which the carbonyl carbon of the substrate is attacked by Zn-hydroxyl (or Zn-water). An unexpected feature of the bound inhibitor, the cis carbamoyl ester bond at the benzyloxycarbonyl linkage to alanine, allows the benzyloxycarbonyl phenyl ring of the inhibitor to interact favorably with Tyr-198. This complex structure is compared with previous structures of carboxypeptidase A, including the complexes with the potato inhibitor, a hydrated keto methylene substrate analogue, and a phosphonamidate inhibitor. Comparisons are also made with the complexes of thermolysin with some phosphonamidate inhibitors.

Alanine↗

PubMed1999

Purification and some properties of camel carboxypeptidase B.

A carboxypeptidase B-like enzyme was purified 116-fold with a recovery of activity of 29% from a crude extract of camel pancreas by a four-step procedure consisting of two anion exchange chromatographies in succession, gel filtration and hydrophobic interaction chromatography. The enzyme was homogeneous on SDS and non-denaturing gel electrophoresis and on gel isoelectric focusing. Its molecular mass was found to be 31.5 kDa and its isoelectric point was estimated as 6.1. It was active towards a number of substrates that are cleaved by carboxypeptidases B from other species and was also susceptible to inhibition by inhibitors of such enzymes. The camel enzyme showed a pH optimum of 8.0 and it was seen to be a relatively potent kininase in vitro. The enzyme purified in this study was very similar to carboxypeptidases B isolated from other species in size, charge, substrate specificity and susceptibility to inhibition and thus it can be identified as camel carboxypeptidase B.

Animals↗

PubMedDate not supplied

Cloning and sequence analysis of cDNA for bovine carboxypeptidase E.

Carboxypeptidase E (enkephalin convertase) was first identified as the carboxypeptidase B-like enzyme involved in the biosynthesis of enkephalin in bovine adrenal chromaffin granules. A similar enzyme is present in many brain regions and in purified secretory granules from rat pituitary and rat insulinoma. Within the secretory granules, carboxypeptidase E (CPE) activity is found in both a soluble and a membrane-bound form, which differ slightly in relative molecular mass (Mr). Here, to investigate whether the CPE activities in the various tissues are produced from a single gene, purified CPE was partially sequenced and oligonucleotide probes were used to isolate a clone encoding CPE from a bovine pituitary complementary DNA library. This cDNA hybridizes to bovine pituitary poly(A)+ RNAs of approximately 3.3, 2.6 and 2.1 kilobases (kb), with the 3.3-kb messenger RNA the predominant species. The predicted amino-acid sequence of the cDNA clone contains the partially determined sequences of CPE, several pairs of basic amino acids and displays some homology with both carboxypeptidases A and B. Restriction analysis of bovine genomic DNA suggests only one gene for CPE. This is consistent with a broad role for CPE in the biosynthesis of many neuropeptides.

Amino Acid Sequence↗

PubMed1993

Characterization of the sporulation-related gamma-D-glutamyl-(L)meso-diaminopimelic-acid-hydrolysing peptidase I of Bacillus sphaericus NCTC 9602 as a member of the metallo(zinc) carboxypeptidase A family. Modular design of the protein.

The sporulation-related gamma-D-glutamyl-(L)meso-diaminopimelic-acid-hydrolysing peptidase I of Bacillus sphaericus NCTC 9602 has been analysed by proton-induced X-ray emission. It contains 1 equivalent Zn2+ per mol of protein. As derived from gene cloning and sequencing, the B. sphaericus Zn peptidase I is a two-module protein. A 100-amino-acid-residue N-terminal domain consisting of two tandem segments of similar sequences, is fused to a 296-amino-acid-residue C-terminal catalytic domain. The catalytic domain belongs to the Zn carboxypeptidase A family, the closest match being observed with the Streptomyces griseus carboxypeptidase [Narahashi (1990) J. Biochem. 107, 879-886] and with the family prototype, bovine carboxypeptidase A. The catalytic domain of the B. sphaericus peptidase I possesses, distributed along the amino-acid sequence, peptide segments, a triad His162-Glu165-His307 and a dyad Tyr347-Glu366 that are equivalent to secondary structures, the zinc-binding triad His69-Glu72-His196 and the catalytic dyad Tyr248-Glu270 of bovine carboxypeptidase A respectively. The N-terminal repeats of the B. sphaericus peptidase I have similarity with the C-terminal repeats of the Enterococcus hirae muramidase 2, the Streptococcus (now Enterococcus) faecalis autolysin and the Bacillus phi PZA and phi 29 lysozymes, to which a role in the recognition of a particular moiety of the bacterial cell envelope has been tentatively assigned. Detergents enhance considerably the specific activity of the B. sphaericus peptidase I.

Amino Acid Sequence↗

PubMed1981

Zinc environment and cis peptide bonds in carboxypeptidase A at 1.75-A resolution.

The structure of the metalloenzyme carboxypeptidase A (peptidyl-L-amino-acid hydrolase, EC 3.4.17.1) has been refined at 1.75 A by a restrained least-squares procedure to a conventional crystallographic R factor of 0.162. Significant results of the refined structure relative to the catalytic mechanism are described. In the native enzyme, the zinc coordination number is five (two imidazole N delta 1 nitrogens, the two carboxylate oxygens of glutamate-72, and a water molecule). In the complex (at 2.0-A resolution) of carboxypeptidase A with the dipeptide glycyl-L-tyrosine, however, the water ligand is replaced by both the carbonyl oxygen and the amino nitrogen of the dipeptide. The amino nitrogen also statistically occupies a second position near glutamate-270. Consequently, the coordination number of zinc may vary from five to six in carboxypeptidase A-substrate complexes. Implications of these results for the catalytic mechanism of carboxypeptidase A are discussed. In addition, three cis peptide bonds, none of which involves proline as the amino nitrogen donor, have been located fairly near the active site.

Animals↗

PubMed1987

Mechanism of carboxypeptidase A: hydration of a ketonic substrate analogue.

The structure of the complex between carboxypeptidase A alpha (EC 3.4.17.1) and the ketonic substrate analogue 5-benzamido-2-benzyl-4-oxopentanoic acid (BOP) has been determined by x-ray crystallographic methods to a resolution of 1.7 A (final R = 0.191). Interestingly, BOP was observed to bind to the active site of carboxypeptidase A alpha as the covalent hydrate adduct. Because BOP is probably less than 0.2% hydrated in aqueous solution, this result was unexpected. One possibility is that the zinc-bound water of the native enzyme added to the ketone carbonyl. Alternatively, the enzyme may preferentially scavenge the hydrated ketone as it is continuously maintained at equilibrium in the solution in which the carboxypeptidase A alpha crystals were immersed. In either case, this mode of binding of BOP to carboxypeptidase A alpha provides an example of the preferred binding of a model of a structure along the reaction coordinate of a hydrolytic reaction.

Carboxypeptidases↗

PubMed1997

Toward antibody-directed enzyme prodrug therapy with the T268G mutant of human carboxypeptidase A1 and novel in vivo stable prodrugs of methotrexate.

Antibody-directed enzyme prodrug therapy (ADEPT) has the potential of greatly enhancing antitumor selectivity of cancer therapy by synthesizing chemotherapeutic agents selectively at tumor sites. This therapy is based upon targeting a prodrug-activating enzyme to a tumor by attaching the enzyme to a tumor-selective antibody and dosing the enzyme-antibody conjugate systemically. After the enzyme-antibody conjugate is localized to the tumor, the prodrug is then also dosed systemically, and the previously targeted enzyme converts it to the active drug selectively at the tumor. Unfortunately, most enzymes capable of this specific, tumor site generation of drugs are foreign to the human body and as such are expected to raise an immune response when injected, which will limit their repeated administration. We reasoned that with the power of crystallography, molecular modeling and site-directed mutagenesis, this problem could be addressed through the development of a human enzyme that is capable of catalyzing a reaction that is otherwise not carried out in the human body. This would then allow use of prodrugs that are otherwise stable in vivo but that are substrates for a tumor-targeted mutant human enzyme. We report here the first test of this concept using the human enzyme carboxypeptidase A1 (hCPA1) and prodrugs of methotrexate (MTX). Based upon a computer model of the human enzyme built from the well known crystal structure of bovine carboxypeptidase A, we have designed and synthesized novel bulky phenylalanine- and tyrosine-based prodrugs of MTX that are metabolically stable in vivo and are not substrates for wild type human carboxypeptidases A. Two of these analogs are MTX-alpha-3-cyclobutylphenylalanine and MTX-alpha-3-cyclopentyltyrosine. Also based upon the computer model, we have designed and produced a mutant of human carboxypeptidase A1, changed at position 268 from the wild type threonine to a glycine (hCPA1-T268G). This novel enzyme is capable of using the in vivo stable prodrugs, which are not substrates for the wild type hCPA1, as efficiently as the wild type hCPA1 uses its best substrates (i.e. MTX-alpha-phenylalanine). Thus, the kcat/Km value for the wild type hCPA1 with MTX-alpha-phenylalanine is 0.44 microM-1 s-1, and kcat/Km values for hCPA1-T268G with MTX-alpha-3-cyclobutylphenylalanine and MTX-alpha-3-cyclopentyltyrosine are 1.8 and 0.16 microM-1 s-1, respectively. The cytotoxic efficiency of hCPA1-268G was tested in an in vitro ADEPT model. For this experiment, hCPA1-T268G was chemically conjugated to ING-1, an antibody that binds to the tumor antigen Ep-Cam, or to Campath-1H, an antibody that binds to the T and B cell antigen CDw52. These conjugates were then incubated with HT-29 human colon adenocarcinoma cells (which express Ep-Cam but not the Campath 1H antigen) followed by incubation of the cells with the in vivo stable prodrugs. The results showed that the targeted ING-1:hCPA1-T268G conjugate produced excellent activation of the MTX prodrugs to kill HT-29 cells as efficiently as MTX itself. By contrast, the enzyme-Campath 1H conjugate was without effect. These data strongly support the feasibility of ADEPT using a mutated human enzyme with a single amino acid change.

Animals↗

PubMed1982

Inhibition of pancreatic carboxypeptidase A: A possible mechanism of interaction between penicillic acid and ochratoxin A.

Penicillic acid and ochratoxin A are environmentally important toxic fungal metabolites (mycotoxins) that are synergistic in combination. The effects of penicillic acid on the pancreatic enzyme, carboxypeptidase A were investigated in vitro and in vivo. A broad range of inhibition in vitro of the enzyme by PA was demonstrated with a half-maximal inhibitory concentration equal to 1.1 x 10(-4) M PA. Inhibition of carboxypeptidase A was time and temperature dependent, and resulted in decreased conversion of parent ochratoxin A to the non-toxic metabolite, ochratoxin alpha. Studies in vivo demonstrated a penicillic acid-dependent inhibition of pancreatic carboxypeptidase A activity in the mouse and the chicken following multiple oral exposure. It is postulated that the mode of toxic interaction of the two mycotoxins may be due, in part, to impaired detoxification of ochratoxin A through penicillic acid depletion of carboxypeptidase A activity.

Animals↗

PubMed1983

Characterization of the carboxypeptidase involved in the proteolytic cleavage of the influenza haemagglutinin.

The arginine carboxypeptidase involved in the proteolytic cleavage of the haemagglutinin of influenza A virus has been analysed by an assay employing a Sepharose-bound peptide containing radioactive arginine as a substrate. The enzyme activity has been extracted from purified virus with non-ionic detergents and has been separated from the haemagglutinin and from the neuraminidase by isoelectric focusing and by affinity chromatography. The carboxypeptidase present in virus grown in different host cells shows variations in its isoelectric point. It can be concluded from these observations that the carboxypeptidase is a host component incorporated into the virus envelope. When the enzyme is inhibited by 2-mercaptomethyl-3-guanidinoethyl-thiopropanoic acid, haemagglutinin with the arginine attached to the carboxy terminus of HA1 can be obtained. The observation that under these conditions the haemagglutinin has retained its haemolytic activity indicates that the carboxypeptidase does not play an essential role in the activation process.

Carboxypeptidases↗

PubMed1982

Tyrosyl interactions at the active site of carboxypeptidase B.

The phosphorescence emission spectra of native carboxypeptidase B and of chemically modified carboxypeptidase B (at arginyl residues) was measured in the presence and absence of peptide and ester substrates (acetyl-L-arginine and its hydroxy ester analog: acetyl-L-argininic acid). Ester binding did not affect the state of the tyrosyl residue as compared with its state in the substrate-free enzyme. In the modified enzyme, which is devoid of peptidase activity, binding of the peptide pseudosubstrate did not perturb the state of the tyrosyl residue. The luminescence spectra of Zn2+- and Co2+-carboxypeptidase B in the presence of the metal coordinating ligand cyanide, used to displace the water from the metal coordination sphere, is also described. Cyanide did not affect the luminescence spectra of the active-site tyrosyl residue in either Zn2+- or Co2+-carboxypeptidase, indicating that the tyrosyl residue was not interacting directly with the metal bound water. Hence, the effect of peptide on tyrosyl phosphorescence is not caused by the displacement of the tyrosyl from the coordination sphere, but rather by direct interaction of the peptide bond. The data are consistent with the proposition that the tyrosyl residue participates as a proton donor in amide but not in ester hydrolysis.

Animals↗

PubMed1980

Purification of two DD-carboxypeptidases/transpeptidases with different penicillin sensitivities from Proteus mirabilis.

Two membrane-bound enzymes of Proteus mirabilis with the dual functions of peptidoglycan DD-carboxypeptidase and transpeptidase (named DD-carboxypeptidase/transpeptidase H and L) were isolated and purified by selective solubilization with the nonionic detergent Genapol X-100, affinity chromatography on matrix-bound ampicillin, and preparative isoelectric focusing in the presence of detergent. Purified enzymes H and L were, respectively, penicillin-binding proteins 4 and 5 among seven major penicillin-binding proteins present in P. mirabilis. The enzymes differed in the following properties. Enzyme H had an Mr of 49,000; isoelectric point at pH 8.2; high sensitivity to benzylpenicillin and permanent inactivation because of high stability of the enzyme-antibiotic complex EI* (half-life 300 min); fragmentation of benzylpenicillin with formation of phenylacetylglycine during the slow decay of EI*; it functioned as an endopeptidase on peptide-crosslinked side chains of peptidoglycan. Enzyme L had an Mr of 43 000; isoelectric point at pH 5.9; low sensitivity to benzylpenicillin and low stability of EI* (half-life 7.2 min) with rapid recovery of enzyme activity; no function as an endopeptidase. The properties of enzyme L were identical with those of the single active DD-carboxypeptidase found previously in the spheroplast L-form of P. mirabilis grown in the presence of benzylpenicillin. We conclude that the partial penicillin resistance of P. mirabilis, with growth as L-form and synthesis of peptide-crosslinked peptidoglycan, depends on the continuing fuction of enzyme L as a DD-carboxypeptidase and transpeptidase in the presence of the antibiotic.

Carboxypeptidases↗