Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.10.3.3 (ascorbate oxidase)
 778 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

(1) 45% of the total copper of green zucchini ascorbate oxidase is EPR-detectable. At least two species of copper are present, one with a small A parallel (Type 1) and one with a large A parallel (Type 2). Computer simulated spectra indicated 50% contribution by each type of copper. (2) Azide inhibited ascorbate oxidase activity by an uncompetitive mechanism. EPR and optical spectra performed on titration of ascorbate oxidase with azide indicated the formation of a copper-azide complex. The Type 2 copper appears to be the binding site of azide. The involvement of the EPR non-detectable copper as an anion binding site with high affinity toward azide can not be excluded.
Mol Cell Biochem 1975 May 30
PMID:The effect of azide on the spectral and catalytic properties of ascorbate oxidase. 16 78

The crystal structure of the fully oxidized form of ascorbate oxidase (EC 1.10.3.3) from Zucchini has been refined at 1.90 A (1 A = 0.1 nm) resolution, using an energy-restrained least-squares refinement procedure. The refined model, which includes 8764 protein atoms, 9 copper atoms and 970 solvent molecules, has a crystallographic R-factor of 20.3% for 85,252 reflections between 8 and 1.90 A resolution. The root-mean-square deviation in bond lengths and bond angles from ideal values is 0.011 A and 2.99 degrees, respectively. The subunits of 552 residues (70,000 Mr) are arranged as tetramers with D2 symmetry. One of the dyads is realized by the crystallographic axis parallel to the c-axis giving one dimer in the asymmetric unit. The dimer related about this crystallographic axis is suggested as the dimer present in solution. Asn92 is the attachment site for one of the two N-linked sugar moieties, which has defined electron density for the N-linked N-acetyl-glucosamine ring. Each subunit is built up by three domains arranged sequentially on the polypeptide chain and tightly associated in space. The folding of all three domains is of a similar beta-barrel type and related to plastocyanin and azurin. An analysis of intra- and intertetramer hydrogen bond and van der Waals interactions is presented. Each subunit has four copper atoms bound as mononuclear and trinuclear species. The mononuclear copper has two histidine, a cysteine and a methionine ligand and represents the type-1 copper. It is located in domain 3. The bond lengths of the type-1 copper centre are comparable to the values for oxidized plastocyanin. The trinuclear cluster has eight histidine ligands symmetrically supplied from domain 1 and 3. It may be subdivided into a pair of copper atoms with histidine ligands whose ligating N-atoms (5 NE2 atoms and one ND1 atom) are arranged trigonal prismatic. The pair is the putative type-3 copper. The remaining copper has two histidine ligands and is the putative spectroscopic type-2 copper. Two oxygen atoms are bound to the trinuclear species as OH- or O2- and bridging the putative type-3 copper pair and as OH- or H2O bound to the putative type-2 copper trans to the copper pair. The bond lengths within the trinuclear copper site are similar to comparable binuclear model compounds. The putative binding site for the reducing substrate is close to the type-1 copper.(ABSTRACT TRUNCATED AT 400 WORDS)
J Mol Biol 1992 Mar 05
PMID:Refined crystal structure of ascorbate oxidase at 1.9 A resolution. 154 98

This report describes the isolation and characterization of a cDNA clone representing a gene specifically expressed in pollen. A cDNA library was constructed against mRNA from mature pollen of Nicotiana tabacum. It was screened differentially against cDNA from mRNA of leaf and of pollen. One clone, NTPc303, was further characterized. On northern blot this clone hybridizes to a transcript 2100 nucleotides in length. NTPc303 is abundant in pollen. Expression of the corresponding gene is restricted to pollen, because no other generative or vegetative tissue contains transcripts hybridizing to NTPc303. Expression of NTP303 is evolutionarily conserved: homologous transcripts are present in pollen from various plant species. The first NTP303 transcripts are detectable on northern blot at the early bi-nucleate stage and accumulate until the pollen has reached maturity. During germination and pollen tube growth in vitro new NTP303 transcripts appear. This transcription has been proved by northern blots as well as by pulse labelling experiments. Nucleotide sequence analysis revealed that NTPc303 has an open reading frame coding for a predicted protein of 62 kDa. This protein shares homology to ascorbate oxidase and other members of the blue copper oxidase family. A possible function for this clone during pollen germination is discussed.
Plant Mol Biol 1992 Apr
PMID:Characterization of a pollen-specific cDNA clone from Nicotiana tabacum expressed during microgametogenesis and germination. 160 Jan 46

On the basis of the spatial structure of ascorbate oxidase [Messerschmidt, A., Rossi, A., Ladenstein, R., Huber, R., Bolognesi, M., Gatti, G., Marchesini, A., Petruzzelli, R. & Finazzi-Agro, A. (1989) J. Mol. Biol. 206, 513-529], an alignment of the amino acid sequence of the related blue oxidases, laccase and ceruloplasmin is proposed. This strongly suggests a three-domain structure for laccase closely related to ascorbate oxidase and a six-domain structure of ceruloplasmin. These domains demonstrate homology with the small blue copper proteins. The relationships suggest that laccase, like ascorbate oxidase, has a mononuclear blue copper in domain 3 and a trinuclear copper between domain 1 and 3 and ceruloplasmin has mononuclear copper ions in domains 2, 4 and 6 and a trinuclear copper between domains 1 and 6.
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PMID:The blue oxidases, ascorbate oxidase, laccase and ceruloplasmin. Modelling and structural relationships. 240 64

Two crystal forms of the multi-copper protein ascorbate oxidase from Zucchini have been analysed at 2.5 A (1 A = 0.1 nm) resolution and a model of the polypeptide chain and the copper ions and their ligands has been built. Crystal forms M2 and M1 contain a dimer of 140,000 Mr and a tetramer of 280,000 Mr, respectively, in the asymmetric unit. The crystallographic analysis proceeded by multiple isomorphous replacement in M2 followed by solvent flattening and averaging about the local dyad axis. M1 was solved by Patterson search techniques using the M2 electron density. M1 was fourfold averaged. M1 and M2 were combined and the process of averaging repeated in cycles. An atomic model was built into the resulting electron density map and refinement initiated. The current R values of M2 and M1 are 24.5% and 32.6%, respectively. Excellent stereo chemistry was maintained, with root-mean-square deviations of bond lengths and bond angles from average values of 0.02 A and 3.1 degrees, respectively. Each subunit of about 550 amino acid residues has a globular shape with dimensions of 49 A x 53 A x 65 A. It is built up by three domains arranged sequentially on the polypeptide chain and tightly associated in space. The folding of all three domains is of a similar beta-barrel type. It is distantly related to plastocyanin. Each subunit has four copper atoms bound as mononuclear and trinuclear species. The mononuclear copper has two histidine, a cysteine, and a methionine ligand and represents the type-1 copper. It is located in the third domain. The trinuclear cluster has eight histidine ligands. It may be subdivided into a pair of copper atoms with six histidine ligands arranged trigonal prismatic. The pair probably represents the type-3 copper. The remaining copper has two histidine ligands. Its third site of co-ordination is formed by the pair of copper atoms. The fourth ligand may be OH- represented by a small protrusion of electron density. This copper probably is the type-2 copper. The symmetry of the trinuclear cluster is C2 and the ligands are supplied symmetrically by domains 1 and 3. However, domain 1 does not contain a type-1 copper and lacks the characteristic ligands. The unprecedented trinuclear cluster probably represents the oxygen binding and electron storage site.
J Mol Biol 1989 Apr 05
PMID:X-ray crystal structure of the blue oxidase ascorbate oxidase from zucchini. Analysis of the polypeptide fold and a model of the copper sites and ligands. 271 59

The copper enzyme ascorbate oxidase, purified from green zucchini squash, has been crystallized at pH 5.4 employing the organic solvent 2-methyl-2,4-pentanediol. The crystals obtained are larger than one millimetre and belong to the space group P2(1)2(1)2, with unit cell parameters; a = 106.7 A, b = 105.1 A, c = 113.5 A. The crystallographic asymmetric unit contains two subunits of the enzyme (Mr = 140,000) and the solvent content of the crystals is 46% (v/v). The diffraction pattern extends to 2.5 A resolution; this crystal form is suitable for a X-ray structural investigation.
J Mol Biol 1983 Sep 05
PMID:A new crystalline modification of the copper enzyme ascorbate oxidase. 662 Mar 82

The optical properties, copper content, catalytic activity and quaternary structure of many preparations of ascorbate oxidase purified with two different methods were examined. Fresh samples appeared identical and were characterized by optical ratios A280/A610 = 25 +/- 1 and A330/A610 = 0.8 +/- 0.05, by specific activity toward ascorbate of 3.48 +/- 0.05 mol g-1 min-1 and by a copper content of 8 +/- 0.3 mol/145 000 Mr. The enzyme is composed of two non-covalently linked subunits of slightly different molecular mass (75 000 and 72 000 respectively). These subunits cannot be further resolved by reduction of disulfide bonds. Proteolytic cleavage of the protein chains was observed during purification and storage in the absence of the protease inhibitor 6-amino caproic acid. Ascorbate oxidase exists as a monomer at neutral pH and undergoes reversible association into higher molecular weight species at slightly acid pH values. Association is not accompanied by spectroscopic or catalytic changes.
Mol Cell Biochem 1983
PMID:A reinvestigation on the quaternary structure of ascorbate oxidase from Cucurbita pepo medullosa. 664 10

The reaction between mushroom tyrosinase and L-ascorbic acid was studied by oxymetric assays and evidence pointing to ascorbate oxidase activity of this enzyme has been obtained. The activity is clearly linear to enzyme concentration and the Michaelis constant for L-ascorbic acid has a value of 2.69 +/- 0.11 mM. Maximum activity is obtained at pH 7.5. A possible reaction mechanism, which is based on the different enzymatic forms of tyrosinase, is also presented.
Biochem Mol Biol Int 1995 Jun
PMID:Mushroom tyrosinase has an ascorbate oxidase activity. 766 34

Detailed investigations of the EPR-active copper ion in the trinuclear type 2/type 3 cluster site of T1Hg Rhus vernicifera laccase suggest that at least some inhibitor anions bind to what was an EPR-silent copper center of the resting enzyme. The key observation is that with [15N]azide the adduct exhibits remarkably well resolved ligand hyperfine structure indicative of splitting from three protein (histidine) nitrogens and one azide nitrogen. This accords nicely with recent X-ray diffraction studies of adducts of the related enzyme, ascorbate oxidase (A. Messerschmidt, H. Luecke, and R. Huber, 1993, J. Mol. Biol. 230, 997-1014). We have also characterized a previously unknown dicyanide adduct that exhibits an EPR signal with ligand hyperfine structure from two protein nitrogens and two cyanide carbons. Cyanide may bind to the same copper center as azide, but not without a structural reorganization of the cluster. The results also imply that the protonation of a bridging ligand within the type 2/type 3 cluster explains the pH dependence of anion binding. Imidazole interacts with the protein but does not bind to the EPR-active copper. In keeping with the function of the dioxygen reduction site, the type 2/type 3 cluster in laccase proves to be an extremely flexible host capable of accommodating a variety of ligands.
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PMID:EPR studies of ligand binding to the type 2/type 3 cluster in tree laccase. 797 82

The X-ray structures of three functional derivatives of ascorbate oxidase (EC 1.10.3.3) from Zucchini have been determined and are compared to the "native" oxidized form. The fully reduced form of ascorbate oxidase has been refined to a crystallographic R-factor of 19.6% for all reflections between 8.0 A and 2.2 A resolution. The geometry at the type-1 copper (CU1) is unchanged compared to the oxidized form, but the oxygen ligand bridging the copper ions CU2 and CU3 (spectroscopic type-3 copper pair) is released and the copper ions move apart yielding a trigonal planar co-ordination with their ligating histidine residues. The co-ordination at the copper ion CU4 (spectroscopic type-2 copper) is not affected. The copper-copper distances increase from an average 3.7 A in the native form to 5.1 A for CU2-CU3, 4.4 A for CU2-CU4 and 4.1 A for CU3-CU4. The peroxide derivative of ascorbate oxidase has been refined to a crystallographic R-factor of 16.0% for all reflections between 8.0 A and 2.59 A resolution. The geometry at the type-1 copper site is not changed compared to the oxidized form. The oxygen ligand bridging copper atoms CU2 and CU3 is lost, too. The peroxide binds terminally to the copper ion CU2 as hydroperoxide. Copper ion CU2 is fourfold co-ordinated to the NE2 atoms of the three histidine residues and to the oxygen atom of the terminally bound peroxide molecule in a distorted tetrahedral geometry. Copper ion CU3 is threefold co-ordinated as in the reduced form and co-ordination around copper atom CU4 is unaltered. The copper-copper distances increase to 4.8 A for CU2-CU3 and 4.5 A for CU2-CU4. The distance CU3-CU4 remains 3.7 A. Treatment with peroxide causes a partial depletion of copper ion CU2. The refinement for the azide derivative of ascorbate oxidase converged at a crystallographic R-factor of 17.8% for all reflections between 8.0 A and 2.32 A. There are no significant structural changes at the type-1 copper site. The oxygen ligand bridging copper ions CU2 and CU3 is again released. Two azide molecules bind terminally to copper ion CU2. Copper ion CU2 is fivefold co-ordinated to the NE2 atoms of the three histidine residues and to both terminally bound azide molecules in a trigonal-bipyramidal manner. Copper-copper distances increase to 5.1 A for CU2-CU3 and 4.6 A for CU2-CU4. The distance CU3-CU4 is decreased to 3.6 A.(ABSTRACT TRUNCATED AT 400 WORDS)
J Mol Biol 1993 Apr 05
PMID:X-ray structures and mechanistic implications of three functional derivatives of ascorbate oxidase from zucchini. Reduced, peroxide and azide forms. 847 45


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