Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A set of stable nitroxide free radicals that are used as spin labels have been shown to possess metal-independent superoxide dismutase-like activity. Unlike superoxide dismutase (SOD), these compounds are low molecular weight, and readily penetrate into the cell. A representative nitroxide, 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy (Tempol), was investigated for antimutagenic activity in the XPRT forward mutation assay in CHO AS52 cells. AS52 cells were exposed to hydrogen peroxide, or the hypoxanthine/xanthine oxidase superoxide generating system, in the presence or absence of 10 mM Tempol. Tempol itself was not mutagenic or toxic to AS52 cells. Tempol protected cells nearly completely from the cytotoxic and mutagenic effects of hydrogen peroxide and hypoxanthine/xanthine oxidase. We have previously shown that nitroxides do not alter the extracellular concentration of hydrogen peroxide, and that they are taken up by mammalian cells, suggesting that the antimutagenic activity of Tempol is an intracellular phenomenon.
Environ Mol Mutagen 1992
PMID:Antimutagenicity of a low molecular weight superoxide dismutase mimic against oxidative mutagens. 131 80

We report here a theoretical study of a series of fentanyl analogs with a wide range of affinities and selectivities at the mu receptor, designed to identify and characterize the molecular determinants of mu receptor recognition. In this work, a complete conformational search combining nested rotations and molecular dynamic simulations has been made, leading to identification of accessible conformers for all analogs and to the selection of a candidate bioactive form. In addition, electronic properties have been calculated and examined as possible modulators of recognition at the mu receptor. The results of these studies have led to a distinct pharmacophore for interaction at the mu receptor for this class of compounds, with the piperidine ring in a chair conformation and the N-phenethyl and 4-phenylpropanamide substituents both equatorial. Moreover, four key moieties necessary for optimum receptor recognition and a postulated role for each of them in this recognition have been identified. These are (i) a protonated amine nitrogen, assumed to be involved in an initial electrostatic interaction with a negatively charged site on the receptor; (ii) a polar function capable of hydrogen-bonding with an electrophilic site; (iii) an aromatic ring involved in lipophilic interaction with a similar moiety; and (iv) a second aromatic ring, most probably involved in electron transfer interaction with the receptor. These requirements, taken together, form the basis of our proposed mechanism for mu receptor recognition. Not only is the presence of these components required for recognition, but specific steric relationships between them have been determined, implying the appropriate arrangement for interaction with complementary receptor sites. These steric parameters are pseudobond angles and one torsion angle that determine the relative spatial arrangement of these four moieties. They are the angles theta 1 and theta 3, defining the relative position of the protonated nitrogen and the polar function with each of the two aromatic rings, and the torsion angle eta 1, defining the orientation of the lone pair(s) on the polar proton-accepting function with respect to the lone pair on the piperidine nitrogen. This postulated mechanism of recognition provides a conceptual framework to understand why some compounds do and some do not recognize the mu receptor.
Mol Pharmacol 1992 Jan
PMID:Molecular determinants of mu receptor recognition for the fentanyl class of compounds. 131 Jan 42

The crystal structure of a mutant ribonuclease T1 (Y45W) complexed with a non-cognizable ribonucleotide, 2'AMP, has been determined and refined to an R-factor of 0.159 using X-ray diffraction data at 1.7 A resolution. A specific complex of the enzyme with 2'GMP was also determined and refined to an R-factor of 0.173 at 1.9 A resolution. The adenine base of 2'AMP was found at a base-binding site that is far apart from the guanine recognition site, where the guanine base of 2'GMP binds. The binding of the adenine base is mediated by a single hydrogen bond and stacking interaction of the base with the imidazole ring of His92. The mode of stacking of the adenine base with His92 is similar to the stacking of the guanine base observed in complexes of ribonuclease T1 with guanylyl-2',5'-guanosine, reported by Koepke et al., and two guanosine bases, reported by Lenz et al., and in the complex of barnase with d(GpC), reported by Baudet & Janin. These observations suggest that the site is non-specific for base binding. The phosphate group of 2'AMP is tightly locked at the catalytic site with seven hydrogen bonds to the enzyme in a similar manner to that of 2'GMP. In addition, two hydrogen bonds are formed between the sugar moiety of 2'AMP and the enzyme. The 2'AMP molecule adopts the anti conformation of the glycosidic bond and C-3'-exo sugar pucker, whereas 2'GMP is in the syn conformation with C-3'-endo-C'-2'-exo pucker. The mutation enhances the binding of 2'GMP with conformational changes of the sugar ring and displacement of the phosphate group towards the interior of the catalytic site from the corresponding position in the wild-type enzyme complex. Comparison of two crystal structures obtained provides a solution to the problem that non-cognizable nucleotides exhibit unexpectedly strong binding to the enzyme, compared with high specificity in nucleolytic activity. The results indicate that the discrimination of the guanine base from the other nucleotide bases at the guanine recognition site is more effective than that estimated from nucleotide-binding experiments so far.
J Mol Biol 1992 Feb 20
PMID:Three-dimensional structure of a mutant ribonuclease T1 (Y45W) complexed with non-cognizable ribonucleotide, 2'AMP, and its comparison with a specific complex with 2'GMP. 131 85

The crystal structure of RNase H from Escherichia coli has been determined by the multiple isomorphous replacement method, and refined by the stereochemically restrained least-squares procedure to a crystallographic R-factor of 0.196 at 1.48 A resolution. In the final structure, the root-mean-square (r.m.s.) deviation for bond lengths is 0.017 A, and for angle distances 0.036 A. The structure is composed of a five-stranded beta-sheet and five alpha-helices, and reveals the details of hydrogen bonding, electrostatic and hydrophobic interactions between intra- and intermolecular residues. The refined structure allows an explanation of the particular interactions between the basic protrusion, consisting of helix alpha III and the following loop, and the remaining major domain. The beta-sheet, alpha II, alpha III and alpha IV form a central hydrophobic cleft that contains all six tryptophan residues, and presumably serves to fix the orientation of the basic protrusion. Two parallel adjacent helices, alpha I and alpha IV, are associated with a few triads of hydrophobic interactions, including many leucine residues, that are similar to the repeated leucine motif. The well-defined electron density map allows detailed discussion of amino acid residues likely to be involved in binding a DNA/RNA hybrid, and construction of a putative model of the enzyme complexed with a DNA/RNA hybrid oligomer. In this model, a protein region, from the Mg(2+)-binding site to the basic protrusion, covers roughly two turns of a DNA/RNA hybrid double helix. A segment (11-23) containing six glycine residues forms a long loop between the beta A and beta B strands. This loop, which protrudes into the solvent region, lies on the interface between the enzyme and a DNA/RNA hybrid in the model of the complex. The mean temperature factors of main-chain atoms show remarkably high values in helix alpha III that constitutes the basic protrusion, suggesting some correlation between its flexibility and the nucleic acid binding function. The Mg(2+)-binding site, surrounded by four invariant acidic residues, can now be described more precisely in conjunction with the catalytic activity. The arrangement of molecules within the crystal appears to be dominated by the cancelling out of a remarkably biased charge distribution on the molecular surface, which is derived in particular from the separation between the acidic Mg(2+)-binding site and the basic protrusion.
J Mol Biol 1992 Feb 20
PMID:Structural details of ribonuclease H from Escherichia coli as refined to an atomic resolution. 131 86

High-resolution three-dimensional structural analyses of yeast iso-1-cytochrome c have now been completed in both oxidation states using isomorphous crystalline material and similar structure determination methodologies. This approach has allowed a comprehensive comparison to be made between these structures and the elucidation of the subtle conformational changes occurring between oxidation states. The structure solution of reduced yeast iso-1-cytochrome c has been published and the determination of the oxidized protein and a comparison of these structures are reported herein. Our data show that oxidation state-dependent changes are expressed for the most part in terms of adjustments to heme structure, movement of internally bound water molecules and segmental thermal parameter changes along the polypeptide chain, rather than as explicit polypeptide chain positional shifts, which are found to be minimal. This result is emphasized by the retention of all main-chain to main-chain hydrogen bond interactions in both oxidation states. Observed thermal factor changes primarily affect four segments of polypeptide chain. Residues 37-39 show less mobility in the oxidized state, with Arg38 and its side-chain being most affected. In contrast, residues 47-59, 65-72 and 81-85 have significantly higher thermal factors, with maximal increases being observed for Asn52, Tyr67 and Phe82. The side-chains of two of these residues are hydrogen bonded to the internally bound water molecule, Wat166, which shows a large 1.7 A displacement towards the positively charged heme iron atom in the oxidized protein. Further analyses suggest that Wat166 is a major factor in stabilizing both oxidation states of the heme through differential orientation of dipole moment, shift in distance to the heme iron atom and alterations in the surrounding hydrogen bonding network. It also seems likely that Wat166 movement leads to the disruption of the hydrogen bond from the side-chain of Tyr67 to the Met80 heme ligand, thereby further stabilizing the positively charged heme iron atom in oxidized cytochrome c. In total, there appear to be three regions about which oxidation state-dependent structural changes are focussed. These include the pyrrole ring A propionate group, Wat166 and the Met80 heme ligand. All three of these foci are linked together by a network of intermediary interactions and are localized to the Met80 ligand side of the heme group. Associated with each is a corresponding nearby segment of polypeptide chain having a substantially higher mobility in the oxidized protein.(ABSTRACT TRUNCATED AT 400 WORDS)
J Mol Biol 1992 Feb 20
PMID:Oxidation state-dependent conformational changes in cytochrome c. 131 91

We have previously demonstrated a reduction in the deformability of neutrophils, exposed to whole particulate cigarette smoke in vitro, by measuring their ability to filter through a micropore membrane with pore dimensions similar to those of the average pulmonary capillary segment. In this study, we exposed neutrophils to the vapor phase of cigarette smoke and investigated the mechanism of the reduction in neutrophil filterability. Although both stimulated neutrophils and smoke-exposed neutrophils demonstrated an increase in filtration pressures, and thus a reduction in cell deformability, compared with control untreated cells, the spontaneous release of the reactive oxygen intermediates hydrogen peroxide and the superoxide anion was depressed following in vitro smoke exposure and there was no shape change to suggest that smoke-exposed cells were activated. The presence of erythrocytes, plasma, or the antioxidants albumin and glutathione prevented the reduction in cell filterability following smoke exposure, suggesting that in vitro smoke exposure, in our system, was mediated by oxidants. Indeed, the increase in filtration pressures, produced by smoke, could be mimicked by the addition of the oxidant hypochlorous acid. The cytoskeletal inhibitors cytochalasin B and D improved the filterability of smoke-exposed cells, suggesting that smoke may change neutrophil deformability through an effect on the actin component of the cytoskeleton. By contrast, colchicine, a specific inhibitor of the microtubules, had no effect. Preincubation with a monoclonal antibody to the CD18 antigen, to block this major neutrophil adhesive glycoprotein, did not alter the filtration pressure developed by stimulated or smoke-exposed neutrophils, suggesting that increased adhesivity was not the mechanism of the increase in filtration pressures observed following smoke exposure.(ABSTRACT TRUNCATED AT 250 WORDS)
Am J Respir Cell Mol Biol 1992 Mar
PMID:Changes in neutrophil deformability following in vitro smoke exposure: mechanism and protection. 131 95

In the genetically mutated ribonuclease T1 His92Ala (RNase T1 His92Ala), deletion of the active site His92 imidazole leads to an inactive enzyme. Attempts to crystallize RNase T1 His92Ala under conditions used for wild-type enzyme failed, and a modified protocol produced two crystal forms, one obtained with polyethylene glycol (PEG), and the other with phosphate as precipitants. Space groups are identical to wild-type RNase T1, P2(1)2(1)2(1), but unit cell dimensions differ significantly, associated with different molecular packings in the crystals; they are a = 31.04 A, b = 62.31 A, c = 43.70 A for PEG-derived crystals and a = 32.76 A, b = 55.13 A, c = 43.29 A for phosphate-derived crystals, compared to a = 48.73 A, b = 46.39 A, c = 41.10 A for uncomplexed wild-type RNase T1. The crystal structures were solved by molecular replacement and refined by stereochemically restrained least-squares methods based on Fo greater than or equal to sigma (Fo) of 3712 reflections in the resolution range 10 to 2.2 A (R = 15.8%) for the PEG-derived crystal and based on Fo greater than or equal to sigma (Fo) of 6258 reflections in the resolution range 10 to 1.8 A (R = 14.8%) for the phosphate-derived crystal. The His92Ala mutation deletes the hydrogen bond His92N epsilon H ... O Asn99 of wild-type RNase T1, thereby inducing structural flexibility and conformational changes in the loop 91 to 101 which is located at the periphery of the globular enzyme. This loop is stabilized in the wild-type protein by two beta-turns of which only one is retained in the crystals obtained with PEG. In the crystals grown with phosphate as precipitant, both beta-turns are deleted and the segment Gly94-Ala95-Ser96-Gly97 is so disordered that it is not seen at all. In addition, the geometry of the guanine binding site in both mutant studies is different from "empty" wild-type RNase T1 but similar to that found in complexes with guanosine derivatives: the Glu46 side-chain carboxylate hydrogen bonds to Tyr42 O eta; water molecules that are present in the guanine binding site of "empty" wild-type RNase T1 are displaced; the Asn43-Asn44 peptide is flipped such that phi/psi-angles of Asn44 are in alpha L-conformation (that is observed in wild-type enzyme when guanine is bound).(ABSTRACT TRUNCATED AT 400 WORDS)
J Mol Biol 1992 Apr 05
PMID:His92Ala mutation in ribonuclease T1 induces segmental flexibility. An X-ray study. 131 2

The purpose of this study was to explore the role of singlet oxygen in cardiovascular injury. To accomplish this objective, we investigated the effect of singlet oxygen [generated from photoactivation of rose-bengal] on the calcium transport and Ca(2+)-ATPase activity of cardiac sarcoplasmic reticulum and compared these results with those obtained by superoxide radical, hydrogen peroxide and hydroxyl radical. Isolated cardiac SR exposed to rose bengal (10 nM) irradiated at (560 nm) produced a significant inhibition of Ca2+ uptake; from 2.27 +/- 0.05 to 0.62 +/- 0.05 mumol Ca2+/mg.min (mean +/- SE) (P less than 0.01) and Ca(2+)-ATPase activity from 2.08 +/- 0.05 mumol Pi/min.mg to 0.28 +/- 0.04 mumol Pi/min.mg (mean +/- SE) (P less than 0.01). The inhibition of calcium uptake and Ca(2+)-ATPase activity by rose bengal derived activated oxygen (singlet oxygen) was dependent on the duration of exposure and intensity of light. The singlet oxygen scavengers ascorbic acid and histidine significantly protected SR Ca(2+)-ATPase against rose bengal derived activated oxygen species but superoxide dismutase and catalase did not attenuate the inhibition. SDS-polyacrylamide gel electrophoresis of SR exposed to photoactivated rose bengal up to 14 min, demonstrated complete loss of Ca(2+)-ATPase monomer band which was significantly protected by histidine. Irradiation of rose bengal also caused an 18% loss of total sulfhydryl groups of SR. On the other hand, superoxide (generated from xanthine oxidase action on xanthine) and hydroxyl radical (0.5 mM H2O2 + Fe(2+)-EDTA) as well as H2O2 (12 mM) were without any effect on the 97,000 dalton Ca(2+)-ATPase band of sarcoplasmic reticulum.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Biochem 1992 Apr
PMID:Singlet oxygen: a potential culprit in myocardial injury? 131 3

The three-dimensional structure of the enzyme myeloperoxidase has been determined by X-ray crystallography to 3 A resolution. Two heavy atom derivatives were used to phase an initial multiple isomorphous replacement map that was subsequently improved by solvent flattening and non-crystallographic symmetry averaging. Crystallographic refinement gave a final model with an R-factor of 0.257. The root-mean-square deviations from ideality for bond lengths and angles were 0.011 A and 3.8 degrees. Two, apparently identical, halves of the molecule are related by local dyad and covalently linked by a single disulfide bridge. Each half-molecule consists of two polypeptide chains of 108 and 466 amino acid residues, a heme prosthetic group, a bound calcium ion and at least three sites of asparagine-linked glycosylation. There are six additional intra-chain disulfide bonds, five in the large polypeptide and one in the small. A central core region that includes the heme binding site is composed of five alpha-helices. Regions of the larger polypeptide surrounding this core are organized into locally folded domains in which the secondary structure is predominantly alpha-helical with very little organized beta-sheet. A proximal ligand to the heme iron atom has been identified as histidine 336, which is in turn hydrogen-bonded to asparagine 421. On the distal side of the heme, histidine 95 and arginine 239 are likely to participate directly in the catalytic mechanism, in a manner analogous to the distal histidine and arginine of the non-homologous enzyme cytochrome c peroxidase. The site of the covalent linkage to the heme has been tentatively identified as glutamate 242, although the chemical nature of the link remains uncertain. The calcium binding site has been located in a loop comprising residues 168 to 174 together with aspartate 96. Myeloperoxidase is a member of a family of homologous mammalian peroxidases that includes thyroid peroxidase, eosinophil peroxidase and lactoperoxidase. The heme environment, defined by our model for myeloperoxidase, appears to be highly conserved in these four mammalian peroxidases. Furthermore, the conservation of all 12 cysteine residues involved in the six intra-chain disulfide bonds and the calcium binding loop suggests that the three-dimensional structures of members of this gene family are likely to be quite similar.
J Mol Biol 1992 Jul 05
PMID:X-ray crystal structure of canine myeloperoxidase at 3 A resolution. 132 Jan 28

The interaction of urea and guanidinium chloride with proteins has been studied calorimetrically by titrating protein solutions with denaturants at various fixed temperatures, and by scanning them with temperature at various fixed concentrations of denaturants. It has been shown that the observed heat effects can be described in terms of a simple binding model with independent and similar binding sites. Using the calorimetric data, the number of apparent binding sites for urea and guanidinium chloride have been estimated for three proteins in their unfolded and native states (ribonuclease A, hen egg white lysozyme and cytochrome c). The intrinsic and total thermodynamic characteristics of their binding (the binding constant, the Gibbs energy, enthalpy, entropy and heat capacity effect of binding) have also been determined. It is found that the binding of urea and guanidinium chloride by protein is accompanied by a significant decrease of enthalpy and entropy. At all concentrations of denaturants the enthalpy term slightly dominates the entropy term in the Gibbs energy function. Correlation analysis of the number of binding sites and structural characteristics of these proteins suggests that the binding sites for urea and guanidinium chloride are likely to be formed by several hydrogen bonding groups. This type of binding of the denaturant molecules should lead to a significant restriction of conformational freedom within the polypeptide chain. This raises a doubt as to whether a polypeptide chain in concentrated solutions of denaturants can be considered as a standard of a random coil conformation.
J Mol Biol 1992 Jul 20
PMID:Protein interactions with urea and guanidinium chloride. A calorimetric study. 132 62


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