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 spin-lattice and spin-spin relaxation rates were measured of the Gly C alpha and Tyr aryl protons of glycyl-L-tyrosine (Gly-Tyr) bound to manganese(II)-substituted carboxypeptidase A (MnCPA) in aqueous solution. The temperature and frequency dependences of the relaxation rates were analyzed using the Solomon-Bloembergen-Morgan equations. The binding modes of MnCPA with Gly-Tyr in solution are different from that of ZnCPA in crystals. 1. Mn(II)-coordinated water of MnCPA is not excluded by the binding of Gly-Tyr substrate molecules. 2. The Gly carbonyl group does not coordinate tightly to the metal ion of MnCPA. The Gly C alpha protons of Gly-Tyr in the productive binding site are appreciably mobile. 3. A non-productive loose binding of another Gly-Tyr molecule is suggested by simulation of the temperature and frequency dependences of the proton relaxation rates.
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PMID:Nuclear magnetic relaxation study on the interaction of glycyl-L-tyrosine with manganese-carboxypeptidase A in solution. 50 May 77

Properties of carboxypeptidase A of cultured skin fibroblasts from control and cystic fibrosis patients were studied using alpha-N-carbobenzoxy-L-glutamyl-L-tyrosine as substrate. Carboxypeptidase A was inhibited by thiomersal, cyanide, iodoacetate and N-ethylmaleimide in a similar manner for control and cystic fibrosis fibroblasts. Both trypsin and dithiothreitol treatment activated the enzyme, but 1,10-phenanthroline inhibited only in the presence of dithiothreitol. Both Zn2+ and Co2+ reversed this inhibition. Trypsin treatment of carboxypeptidase A produced a form of the enzyme having a higher KM value for both control and cystic fibrosis fibroblasts. Dithiothreitol treatment of control fibroblasts resulted in a form with similar properties to the trypsin activated form, but cystic fibrosis fibroblasts yielded a variant form with even higher KM and Vmax values. Since other properties were similar, it seems likely that this difference reflected binding of a molecule to the enzyme rather than of a defect in the enzyme.
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PMID:Carboxypeptidase A activity of cultured skin fibroblasts and relationship to cystic fibrosis. 66 47

In porcine pancreatic secretion procarboxypeptidase A exists in two states: as a monomer and as a binary complex of a type hitherto not observed in the pancreatic secretions of other species. This complex is shown to contain 1 molecule of procarboxypeptidase A and 1 molecule of a proteolytic zymogen we have designated as zymogen E. The two subunits of the complex have been separated by gel filtration in a denaturing solvent and the products used for compositional and NH2-terminal sequence analysis. We also fractionated the mixture obtained on activation of the binary complex and isolated homogenous preparations of porcine carboxypeptidase A and the diisopropylphosphoryl derivative of the enzyme formed from zymogen E. Zymogen E has an Mr of about 26,000 and on activation produces an enzyme of essentially the same Mr with properties very similar to those of human pancreatic protease E. It catalyzes the esterolysis of acetyl-L-alanyl-L-analyl-L-alanine methyl ester, but is inert towards acetyl-L-tyrosine ethyl ester and is readily inactivated by diisopropylophosphorofluoridate. Zymogen E has an amino acid composition different from those of porcine chymotrypsinogen A, B, or C. Its NH2-terminal sequence shows homology with the NH2-terminal sequence of lungfish proelastase A; yet, like human protease E, porcine protease E has relatively very low activity on intact elastin.
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PMID:Identification of a binary complex of procarboxypeptidase A and a precursor of protease E in porcine pancreatic secretion. 67 Feb 12

Although optical resolution of alpha-methylphenylalanine (alpha-Me-Phe) has been achieved by the action of carboxypeptidase A on the N-trifluoroacetyl derivative of the amino acid (TFA-alpha-Me-Phe), it is improbable that an alpha-methyl substrate could bind in the same orientation as glycyl-L-tyrosine, due to steric interaction of the alpha-methyl group with an atom in the imidazole ring of zinc ligand His-196. The kinetic parameters for TFA-alpha-Me-Phe and for an ester substrate bearing an alpha-methyl group (beta-hippuryl-alpha-methylphenyllactic acid, HMPL) have been determined and compared to those for the appropriate nonmethylated control substrates. Both TFA-alpha-Me-Phe and HMPL appear to be bound nearly as well as are their respective controls, and HMPL is hydrolyzed nearly as rapidly as its control. TFA-alpha-Me-Phe, however, is hydrolyzed only about one-fiftieth as rapidly as is the nonmethylated substrate. These findings are consistent with the possibilities that: (1) the proposed substrate-induced conformational shift of Tyr-248 is hindered when the methylated substrates are bound; (2) the orientation in which alpha-methyl substrates are bound precludes optimal positioning of Tyr-248 and the scissile bond even after the rotation of Tyr-248 has occurred; (3) amides and esters are bound in different orientations, and in the amide orientation an alpha-methyl group is so directed as to interfere with the approach of Glu-270 to the scissile bond.
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PMID:alpha-Methyl substrates of carboxypeptidase A. A steric probe of the active site. 114 72

To find a possible explanation for the selective hepatic conjugation of bile acids with glycine or taurine, the N-acyl amidates of cholic acid and a number of amino acids and amino acid analogues were synthesized, and their susceptibility to hydrolysis by pancreatic juice, gastric juice, serum, or small intestinal mucosal enzymes was measured. Deconjugation by pure carboxypeptidase A and B was also examined, and hydrolysis by these tissue fluids and enzymes was compared with that mediated by a bacterial cholylglycine hydrolase. Human pancreatic juice efficiently hydrolyzed cholyl conjugates of all neutral-L-amino acids (cholyl-L-alanine, cholyl-L-valine, cholyl-L-leucine, and cholyl-L-tyrosine), except cholylglycine. The net hourly rate of hydrolysis (in micromoles per milligram protein per hour) increased when the terminal residue was aromatic or branched aliphatic, and appeared to be specific for L-alpha-amino acids as cholyl-beta-alanine and cholyl-D-valine were not cleaved. From cholyl glycylglycine, only the terminal glycine was efficiently removed. Cholyltaurine and cholyl conjugates with the methyl and propyl analogues of taurine were resistant to hydrolysis. Two basic amino acid conjugates (cholyl-L-lysine and cholyl-L-arginine) were cleaved, whereas conjugates of acidic amino acids (cholyl-aspartate and cholyl-cysteate) were not cleaved. Studies using pure enzymes showed that bovine carboxypeptidase A hydrolyzed the cholyl conjugates of the neutral L-alpha-amino acids with similar specificity as observed for the human pancreatic juice, whereas bovine carboxypeptidase B cleaved the basic amino acid conjugates. Cholyl-L-lysine and cholyl-L-arginine were also cleaved by serum and plasma, which are known to possess carboxypeptidase activity. Cholyl conjugates were not cleaved by gastric juice, by trypsin, or by homogenates of rat small intestinal mucosa. In contrast, all cholyl conjugates were cleaved by a bacterial cholylglycine hydrolase. These experiments indicate that glycine and taurine amidates of cholic acid differ from a number of other conjugates with neutral and basic amino acid in being resistant to hydrolysis by pancreatic and plasma carboxypeptidases.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Pancreatic carboxypeptidase hydrolysis of bile acid-amino conjugates: selective resistance of glycine and taurine amidates. 286

A high-resolution x-ray crystallographic investigation of the complex between carboxypeptidase A (CPA; peptidyl-L-amino-acid hydrolase, EC 3.4.17.1) and the slowly hydrolyzed substrate glycyl-L-tyrosine was done at -9 degrees C. Although this enzyme-substrate complex has been the subject of earlier crystallographic investigation, a higher resolution electron-density map of the complex with greater occupancy of the substrate was desired. All crystal chemistry (i.e., crystal soaking and x-ray data collection) was performed on a diffractometer-mounted flow cell, in which the crystal was immobilized. The x-ray data to 1.6-A resolution have yielded a well-resolved structure in which the zinc ion of the active site is five-coordinate: three enzyme residues (glutamate-72, histidine-69, and histidine-196) and the carbonyl oxygen and amino terminus of glycyl-L-tyrosine complete the coordination polyhedron of the metal. These results confirm that this substrate may be bound in a nonproductive manner, because the hydrolytically important zinc-bound water has been displaced and excluded from the active site. It is likely that all dipeptide substrates of carboxypeptidase A that carry an unprotected amino terminus are poor substrates because of such favorable bidentate coordination to the metal ion of the active site.
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PMID:X-ray crystallographic investigation of substrate binding to carboxypeptidase A at subzero temperature. 346 86

The catalytic properties of a sheep mast cell proteinase (SMCP), isolated from abomasal mucosal mast cells, were investigated. The enzyme was shown to have chymotrypsin-like esterase activity, with no detectable amide activity, using a range of low molecular weight substrates. Maximal activity, against Benzyloxycarbonyl-L-tyrosine-4-nitrophenol ester, was determined to be in the range pH 7.6-8.0. Inhibitor studies showed that, unlike chymotrypsin, a serine proteinase, SMCP was found to be susceptible to the action of thiol blocking agents and chelating agents, but to be unaffected by diisopropylphosphofluoridate, a serine proteinase inhibitor.
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PMID:The catalytic properties of a proteinase isolated from sheep abomasal mucosal mast cells. 353 58

Spectrochemical probes have demonstrated that the conformations of carboxypeptidase A (EC 3.4.12.2) differ in solution and in the crystalline state. Detailed kinetic studies of carboxypeptidase A(alpha) and A(gamma) crystals and solutions now show that the physical state of the enzyme is also a critical parameter that affects the function of the A(alpha) and A(gamma) enzymes in the same manner. The kinetic profiles and the corresponding kinetic constants of substrate hydrolysis are, therefore, important functional indices of the known conformational differences of the enzyme in these two physical states. The complex kinetic behavior of this enzyme, however, precludes meaningful comparisons of activity measurements for crystals and solutions obtained at only one substrate concentration. Underlying differences in varying substrate-inhibiting or -activating binding modes can result in either high or low activity ratios, concealing the true, functional consequences of the change in physical state. Thus, for all substrates examined, crystallization of the enzyme markedly reduces catalytic efficiency, k(cat), from 20- to 1000-fold. Equally as important, the substrate inhibition, apparent in solution for some di- and depsipeptides, is abolished with crystals, while for longer substrates the normal solution kinetics may acquire activation with the crystals. Hypothetical modes of substrate-enzyme interaction, generated by superimposing substrate models on the crystal structure of carboxypeptidase to simulate kinetics in solution, have failed to detect both of these changes, which affect inhibitory or activating binding modes. The only structure of carboxypeptidase yet published and that of its functionally inert complex with the pseudosubstrate, glycyl-L-tyrosine, derive from a unique form of carboxypeptidase A(alpha) crystals. These crystals differ from all others with regard both to their spectral properties and activity toward carbobenzoxy-glycyl-L-phenylalanine, which is 30% of that in solution, though the significance of this value cannot be gauged without knowledge of the relevant kinetic constants. The rapidly accumulating evidence for functional and conformational differences between crystals and solutions and the recent stress on the nonproductive aspects of the carboxypeptidase A(alpha)-glycyl-L-tyrosine complex, based on 30% site occupancy, suggest that the functional implications of its structural features require reevaluation.
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PMID:The physical state dependence of carboxypeptidase Aalpha and Agamma kinetics. 453 Feb 72

Stimulation of rat mast cell suspension from actively sensitized rats with antigen in vitro produced a parallel release of histamine and enzyme, probably proteolytic activity, which releases p-nitrophenol from an L-tyrosine-p-nitrophenyl ester derivative (TPNE). The histamine and enzyme release correlated with respect to their dependence on antigen concentration, reaction time and inhibition by 2,4-DNP and papaverine. In contrast, more than 50% of total histamine but nearly no enzyme was released by the ionophore A 23.187 and C 48/80 (each less than or equal to 1 microgram/ml). The enzyme was apparently secreted predominantly in a particular form. It was approximately 50% inactivated by heating for 1 h at 56 degree C or by incubation for 3 h at 37 degrees C with the chymotrypsin inactivator tosyl-phenylalanine chloromethylketone (TPCK; 2.5 X 10-4 M) or for 5 min at 37 degrees C with benzyl sulphonyl fluoride (2.5 X10-4 M), which reacts with SH groups. Heating for 3 min at 100 degrees C destroyed it completely. On the basis of these properties we suggest that the antigen released enzyme is the known granulabound chymase from rat mast cells. TPNE was not only a cleavable substrate for the enzymatic activity in the 800 g cell supernatant following antigen stimulation, but also a strong inhibitor of the histamine release on administration before antigen (IC50 approximately 10-6 M). It appears that the same enzyme activity acts initially intracellularly as activator of the histamine secretion and then is subsequently released along with histamine as a further mediator. Extracellularly this enzyme may act as a modulator of inflammatory reactions in type I allergy both locally and systemically.
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PMID:Selective antigen-stimulated release of proteolytic activity from rat mast cells. 616 86

The catalytic role of the metal ion in bovine carboxypeptidase A (peptidyl-L-amino acid hydrolase; EC 3.4.12.2) is investigated by application of cryoenzymologic and electron paramagnetic resonance methods with use of the Co2+-reconstituted enzyme. Incorporation of 17O into oxygen-donor ligands induces a substantial change in the spin-lattice relaxation probability of the paramagnetic ion. While a change in spin-lattice relaxation is observed for the free Co2+-enzyme in 17O-enriched water, no change is observed for the enzyme complexed to glycyl-L-tyrosine. These results are consistent with x-ray crystallographic studies showing that the metal-bound water molecule in the active site is displaced upon binding of the peptide inhibitor. A change in spin-lattice relaxation of the Co2+ ion in the mixed anhydride, acyl-enzyme intermediate formed with the specific ester substrate O-(trans-p-chlorocinnamoyl)-L-beta-phenyllactate is observed when 17O is enriched either into water or into the carbonyl oxygen position of the scissile bond of the substrate. Since the protein supplies three amino acid side chains as ligands to the metal ion, these results indicate that the metal ion is altered from a tetra-coordinate species in the free enzyme to a penta-coordinate species in the acyl-enzyme reaction intermediate. In addition, the results provide structural support for our assignment of ionization of a metal-bound water molecule in rate-limiting deacylation (Makinen, M. W., Kuo, L. C., Dymowski, J. J., and Jaffer, S. (1979) J. Biol. Chem. 254, 356-366) and affirm that the metal-hydroxide species is the nucleophile responsible for the breakdown of the mixed anhydride reaction intermediate of carboxypeptidase A.
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PMID:Hydrolysis of esters by carboxypeptidase A requires a penta-coordinate metal ion. 627 27


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