Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P15088 (mast cell)
 14,925 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Refined crystal structure of carboxypeptidase A at 1.54 A resolution. 688 46

4-Dimethylaminophenol, after i.v. injection, rapidly forms ferrihemoglobin and has been successfully used in the treatment of cyanide poisoning. The catalytic ferrihemoglobin formation is terminated by thioether formation of oxidized 4-dimethylaminophenol with reduced glutathione or cysteine 93 beta of hemoglobin. Hereby the physiological functions of human hemoglobin are markedly altered. After binding of two molecules of 4-dimethylaminophenol to tetrameric hemoglobin, the rate of autoxidation is increased about 6-fold. The oxygen affinity is 10 times higher than normal, the Hill coefficient is diminished nearly to unity, and the Bohr effect is reduced by about 50%. The physiologically important allosteric regulation of the oxygen affinity by 2,3-diphosphoglycerate is abolished, and the binding of 2,3-diphosphoglycerate to deoxyhemoglobin no longer functions. By molecular sieving, two alkylated hemoglobins were separated: a hemoglobin fraction with an unchanged low tetramer dimer dissociation, normal electronic spectra, and normal digestibility by carboxypeptidase A; and a second fraction with a high degree of dissociation, altered electronic spectra, and impaired digestibility. A tryptic peptide was isolated containing cysteine 93 beta and histidine 146 beta cross-linked by an arylic compound missing the dimethylamine label. The following reaction mechanism is concluded: Oxy-hemoglobin catalyzes the oxidation of 4-dimethylaminophenol, and the oxidation product, presumably N,N-dimethylquinonimine, is bound covalently to cysteine 93 beta by a thioether linkage. This adduct is unstable and autoxidizes further with the liberation of dimethylamine. The resulting quinoid thioether electrophilically attacks the COOH-terminal histidine of the beta-chain, thereby forming an intramolecular cross-link. By this latter reaction, hemoglobin lacks allosteric transition upon ligation and is obviously frozen in its quaternary R-state.
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PMID:Site and mechanism of covalent binding of 4-dimethylaminophenol to human hemoglobin, and its implications to the functional properties. 688 71

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.
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PMID:Zinc environment and cis peptide bonds in carboxypeptidase A at 1.75-A resolution. 694 49

We compare the detailed binding modes of the 39-amino acid inhibitor from potatoes, glycyl-L-tyrosine, the ester analogue CH3OC6H4(CO)CH2CH(CO2(-))C6H5, and indole acetate to the exopeptidase carboxypeptidase A (EC 3.4.17.1). In the potato inhibitor, cleavage of the COOH-terminal glycine-39 leaves a new carboxylate anion of valine-38 having one oxygen on zinc and the other as a receptor of a hydrogen bond from tyrosine-248 of carboxypeptidase. Tyrosine-248 also receives a hydrogen bond from the amide proton of the originally penultimate peptide bond between tyrosine-37 and valine-38. This hydrogen bond suggests product stabilization which is available to peptides and depsipeptides but not to esters lacking an equivalent peptide bond (nonspecific esters). Also, this structure may represent the intermediate binding step for the uncleaved substrate as it moves along the binding subsites. In particular, this may be the binding mode for the substrate after association of the COOH-terminal region of the substrate with the residues at binding subsite S2 (tyrosine-198, phenylalanine-279, and arginine-71) and preceding entry into the catalytic site S1'. These stabilized complexes allow some understanding of the effect of indole acetate, shown here to bind in the pocket at S1', as a competitive inhibitor for esters (for which entry into S1' precedes the rate-determining catalytic step for hydrolysis) and as a noncompetitive inhibitor for peptides (for which entry into S1' is rate limiting). These results, including the binding mode of the ester analogue, are consistent with the original proposal from x-ray studies that both esters and peptides are cleaved with the carboxy terminus at S1', although not necessarily by the same chemical steps.
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PMID:Binding of ligands to the active site of carboxypeptidase A. 694 83

Steady-state and lifetime-resolved fluorescence anisotropy measurements of protein fluorescence were used to investigate the depolarizing motions of tryptophan residues in proteins. Lifetime resolution was achieved by oxygen quenching. The proteins investigated were carbonic anhydrase, carboxypeptidase A, alpha-chymotrypsin, trypsin, pepsin, and bovine and human serum albumin. When corrected for overall protein rotation, the steady state anisotropies indicate that, on the average, the tryptophan residues in these proteins rotate 29 degrees +/- 6 degrees during the unquenched excited state lifetimes of these proteins, which range from 1.7 to 6.1 ns. The lifetime-resolved anisotropies reveal correlation times for these displacements ranging from 1 to 12 ns. On the average these correlation times are tenfold shorter than that expected for overall protein rotation. We conclude that the tryptophan residues in these proteins display remarkable freedom of motion within the protein matrix, which implies that these matrices are highly flexible on the nanosecond time scale.
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PMID:Nanosecond segmental mobilities of tryptophan residues in proteins observed by lifetime-resolved fluorescence anisotropies. 724 63

The production of reactive oxygen intermediates (ROI) in compound 48/80- or calcium ionophore A 23187- activated pleural or peritoneal mast cells was monitored using flow cytometry and the fluorescence indicator dihydrorhodamine 123 (a derivate of rhodamine 123). Mast cell degranulation and ROI production were estimated by flow cytometric fluorescence and light-scatter analysis. In addition, activation was monitored by spectrofluorimetric measurement of histamine secretion from the cells. We used flow cytometric measurement of narrow and wide angle scatter and fluorescence intensity to distinguish between degranulated and resting mast cells during activation and to monitor the two populations separately. Both stimuli induced a dose-dependent elevation of ROI production in mast cells which was accompanied by cell degranulation and histamine secretion. These alterations were completely abolished by preincubation of the mast cells with diethyldithiocarbamate (DTC). Using low concentrations of DTC partial decrease of degranulation and histamine secretion was observed while ROI production was blocked. D-mannitol increased ROI production in stimulated mast cells without a marked effect on degranulation or histamine secretion. The data suggest that mast cell degranulation (histamine secretion) and ROI production are independent processes.
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PMID:Flow cytometric measurement of the production of reactive oxygen intermediate in activated rat mast cells. 752 Jul 10

An MD simulation of the system carboxypeptidase A (CPA) with the tetrapeptide Val-Leu-Phe-Phe has been performed in order to learn about the substrate disposition just prior to nucleophilic attack. We have explored the model in which the substrate does not substitute the zinc-coordinated water (the "water" mechanism). The simulations do suggest as feasible that the Zn-OH2 group performs a nucleophilic attack on the Phe-Phe peptidic bond. We have also investigated the model in which the carbonyl oxygen displaces the zinc-coordinated water. In this case the substrate and Glu-270 orient themselves to allow an anhydride intermediate during the peptidic bond cleavage (the "anhydride" mechanism). Based on the results of the simulations, both "water" and "anhydride" mechanisms are structurally feasible, although the former model seems more probable on chemical grounds.
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PMID:The enzymatic mechanism of carboxypeptidase: a molecular dynamics study. 815 67

X-ray absorption fine structure (XAFS) spectra of carboxypeptidase A (ZnCPD) show progressive spectral changes particularly in the near edge region when the pH is changed from neutral to alkaline values. Both least square fitting and radial distribution function (RDF) data analysis yield two distributions of atoms in the first coordination shell of ZnCPD at all pH values and at both 150 and 297 K. Direct comparison of the first and higher coordination shells of ZnCPD reveals structural differences between pH 7.0 and pH 9.9. At pH 7.0, the zinc ion has four ligand atoms (N or O) at an average distance of 2.024 +/- 0.006 A, and a smaller distribution of 1.3 atoms (N or O) at 2.54 +/- 0.05 A from the zinc ion (from the least square fitting analysis). At pH 9.9, the larger distribution contains four atoms at a 0.022 A shorter distance (2.002 A) from the zinc, while the smaller distribution contains 0.7 atoms at 2.52 +/- 0.06 A. The smaller distribution can be attributed mainly to the contribution of the epsilon 2-oxygen of Glu 72 and to the atoms farther away for which the contribution cannot be fully separated from the first shell peak. The structural changes of ZnCPD at intermediate pHs are consistent with the changes observed at pH 7.0 and pH 9.9. The XAFS Debye-Waller factor shows an increased structural disorder for the four atom distribution at the alkaline pH.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:XAFS studies of carboxypeptidase A: detection of a structural alteration in the zinc coordination sphere coupled to the catalytically important alkaline pKa. 826 59

Competitive inhibition constants Ki for a series of phenol-ring-substituted derivatives of alpha-(2-hydroxyphenyl)benzenepropanoic acid have been ascertained by observing their influence on the catalytic hydrolysis of a peptide substrate by the zinc enzyme carboxypeptidase A. The pH-dependence of Ki shows that binding is maximal between two pKa values: one is that of the phenol group of the inhibitor, and the other uniformly has a value of 6, the pKa of a Zn(2+)-bound water molecule on the enzyme in the absence of substrate or inhibitor. This is the dependence expected if phenolate binds to the Zn2+ displacing its bound H2O/HO-. A log-log plot of the dissociation constants for the productive forms of inhibitor plus enzyme versus the acid dissociation constants of the phenolic residues in the inhibitors yields a straight line with a slope of +0.76. This number indicates that the active-site metal ion has special capacity for dispersing negative charge, such as builds up on the oxygen atom of a carboxamide group undergoing nucleophilic addition.
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PMID:Fluxionate Lewis acidity of the Zn2+ ion in carboxypeptidase A. 842 57

Adenosine, a vasodilator metabolite, is often produced in tissues where the demand for oxygen exceeds the supply. We have recently demonstrated in isolated cannulated arterioles that adenosine and its metabolite, inosine, can also cause vasoconstriction by stimulation of mast cells. Secondary release of histamine and thromboxane is responsible for the inosine-induced constriction in vivo. In the present study, we explored the vasomotor effects of adenosine in vivo and investigated the role of the A3 adenosine receptor in mediating vasoconstriction. In vivo, local application of adenosine (10-6 to 10-4 mol/L) to arterioles consistently caused dose-dependent vasodilation. A fraction of arterioles, however, exhibited a biphasic response, with constriction following dilation. This, too, was dose dependent; 37% of arterioles constricted by 12.7 +/- 4.3% of the initial diameter in response to 10-4 mol/L adenosine. In the presence of 8-(p-sulfophenyl)theophylline (8-SPT), an antagonist of A1 and A2 adenosine receptors, dilation in response to the same dose of adenosine was reduced, and constriction was enhanced; 85% of the tested arterioles constricted by -44.3 +/- 6.0% of the initial diameter. The A3 adenosine receptor has been shown to facilitate mediator release from mast cells, and its role was also examined. N6-(3-Iodo-4-aminobenzyl)adenosine (I-ABA), an agonist of A1 and A3 adenosine receptors, produced dose-dependent vasoconstriction. 1,3-Dipropyl-8-(4-acrylate)phenylxanthine (BW-A1433), an antagonist of A1, A2, and A3 receptors, significantly reduced the vasoconstrictor response to adenosine, which was unmasked during treatment with 8-SPT. In addition, both adenosine and I-ABA stimulated mast cell uptake of ruthenium red, indicating degranulation. The I-ABA-induced constriction was abolished by combined histamine and thromboxane receptor antagonists. We conclude that adenosine can cause vasoconstriction in vivo, which is often masked by A2 receptor-mediated vasodilation. Mast cells are stimulated in the course of the response, and the A3 adenosine receptor is involved in mediating constriction.
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PMID:Adenosine-induced vasoconstriction in vivo. Role of the mast cell and A3 adenosine receptor. 863 20


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