Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:Q9UMR3 (NMR)
 150,598 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The binding of formate to bovine Cu,Zn superoxide dismutase has been studied by NMR spectroscopy. The distance between the copper ion and the proton covalently bound to formate has been evaluated from the broadening of the resonance of such proton. The effect on the copper-coordinated water molecule was evaluated from the bulk water relaxation effect by pulsed low-resolution NMR. The broadening of the resonance due to the formate carboxyl in the 13C NMR spectrum gave further indications about the carbon-copper distance thus providing information about the orientation of the formate ion. Changes of isotropically shifted resonances of the Cu,Co enzyme, where cobalt substitutes the native zinc, indicate that rearrangements of imidazoles of the liganding histidines occur upon binding. Transient NOE experiments gave indication of the proximity of the formate proton to resonance H of the NMR spectrum assigned to the imidazole proton of the copper-liganding His 118 of the active site. 2D NMR NOESY experiments made clear that no important rearrangement of the liganding histidines occurred in the presence of a saturating amount of formate. The absence of relevant changes of the intensity of NOE cross-peaks which are sensitive to interatomic distances in the active site revealed that only slight changes have occurred. Molecular graphics representation on the basis of all the information obtained allowed us to locate the formate in the proximity of the active site. The formate binding occurs via hydrogen bonds through the carboxylate ion and the NH groups of the side chains of Arg 141 which is external to the copper coordination sphere and faces the active site of the enzyme.
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PMID:Formate as an NMR probe of anion binding to Cu,Zn and Cu,Co bovine erythrocyte superoxide dismutases. 146 27

A method for copper- and manganese-containing superoxide dismutase (Cu- and MnSOD) assay in tissue homogenates such as liver and brain, based on the measurement of the longitudinal nuclear relaxation time (T1) of F-, has been developed as a preliminary approach to in vivo measurement of these enzymes. The relaxation rate of F-, which increases linearly with the SOD concentration, also depends on the oxidation state of the metal ion present in the active site of the enzyme. The relaxivity values of the oxidized, reduced and turnovering CuSOD were found to be 9.6 x 10(6), much less than 1 x 10(2) and 5.2 x 10(6) M-1 s-1, respectively, while for MnSOD the corresponding values were 2.9 x 10(6), 4.2 x 10(6) and 3.6 x 10(6) M-1 s-1, respectively. These high relaxivity values allow the detection of SODs in brain and liver homogenates where, under aerobic conditions, these enzymes appear in the steady-state. The contribution of the two types of SOD to the F- relaxation rate in the homogenates was measured by addition of either diethyldithiocarbamate or cyanide, both of which selectively inhibit the CuSOD. The comparison between NMR and activity data confirmed the possibility of carrying out accurate and precise measurements of SODs in homogenates by NMR.
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PMID:NMR method for superoxide dismutase assay in brain and liver homogenates. 164 13

The structure-function relationship in Cu,Zn superoxide dismutase has been partially elucidated by the combined use of many spectroscopic techniques (electronic spectroscopy, circular dichroism, EPR and NMR) and site-directed mutagenesis techniques. The comparison of the spectroscopic and catalytic properties of various mutants, in which some active site residues have been substituted through site-directed mutagenesis, allowed us to establish that the activity is in general more sensitive to electrostatic effects rather than to steric effects or changes in the copper hydration or coordination geometry.
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PMID:Advances in the understanding of the structure-function relationship in Cu,Zn superoxide dismutase. 164 93

The reaction of the Cu,Co derivative of bovine Cu,Zn superoxide dismutase with phenylglyoxal or butanedione, which are known to inactivate the enzyme by selectively binding to Arg 141, has been studied by 1H NMR. Several 1H NMR lines of the copper-liganding histidine residues were perturbed, reproducing an effect so far observed only in the case of binding of anions to this protein. The room temperature EPR spectrum of the modified Cu,Zn protein was altered very slightly, indicating that the geometry of the copper site was not grossly affected by the modification. NMR and EPR changes were reversed by dialysis in the case of the reversible butanedione adduct. These data show that the coordination of the copper in Cu,Zn superoxide dismutase can be destabilized by modifications occurring at a neighboring but not a metal-liganding residue. It is suggested that part of the NMR effects seen on copper ligands in the case of anion binding are produced by interaction of anions with Arg 141, rather than by direct ligand replacement.
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PMID:NMR evidence for perturbation of the copper coordination sphere upon chemical modification of arginine 141 in bovine Cu,Zn superoxide dismutase. 165 80

Human copper-cobalt superoxide dismutase in the reduced form has been investigated through 1H NMR techniques. The aim is to monitor the structural properties of this derivative and to compare them with those of reduced and oxidized native superoxide dismutases. The observed signals of the cobalt ligands have been assigned as well as the signals of the histidines bound to copper(I). The latter signals experience little pseudocontact shifts which allow a rough orientation of the magnetic susceptibility tensor in the molecular frame. The connectivities indicate that, although the histidine bridge is broken in the reduced form, the interproton distances between ligands of both ions are essentially the same.
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PMID:1H NMR investigation of reduced copper-cobalt superoxide dismutase. 178 8

Oxygen radical toxicity has been implicated in the pathogenesis of myocardial reperfusion injury. In the present study we sought to document the existence of a precise temporal relationship between the time course of free radical generation and the time course of alterations of myocardial energy metabolism during early reperfusion. Rabbit hearts perfused within the bore of a 31-Phosphorous NMR spectrometer were subjected to 30 min of total global ischemia at 37 degrees C. At reflow, 12 control hearts received a bolus of normal perfusate and 12 hearts recombinant human superoxide dismutase (h-SOD) as a 60,000 IU bolus followed by a 100 IU/ml infusion for 15 min. Ischemia resulted in similar depletion of tissue ATP and phosphocreatine (PCr) in the two groups. During the first minute of reflow, recovery of PCr was similar in both groups. However, PCr recovery arrested in control hearts after 2 min, at 63% of baseline, and averaged 64 +/- 4% after 45 min of reperfusion. In contrast, h-SOD treated hearts recovered 86.7% of baseline PCr content after 2 min, 102% after 10 min of reperfusion (P less than 0.001), and 93 +/- 6.4% at the end of the 45 min of reflow (P less than 0.01). The time course of free radical formation during reperfusion was assessed by EPR spectroscopy using both the frozen tissue and the spin trapping methodologies. In control hearts, peak generation of oxygen radicals was reached after 20 s of reflow. h-SOD treatment decreased concentrations of the oxygen-centered radicals in myocardial tissue and of the radical-adducts in the coronary effluent by approximately 80%. Thus, in reperfused hearts peak oxygen radical generation is followed by the occurrence of alterations in the recovery of high energy phosphate metabolism. Both events were largely prevented by administration of h-SOD at reflow. These results provide strong support for a link between oxygen free radical generation and post-ischemic reperfusion injury.
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PMID:The relationship between oxygen radical generation and impairment of myocardial energy metabolism following post-ischemic reperfusion. 181 Oct 55

Substitution of the completely conserved aspartic acid residue at position 124 of Cu,Zn superoxide dismutase with asparagine and glycine has been performed through site-directed mutagenesis on the human enzyme. Asp124 is H-bonded to the NH of two histidines, one of which is bound to copper and the other to zinc. The mutant proteins, as expressed in Escherichia coli, result in an essential zinc-free enzyme which is similar to that obtained from the wild-type derivative through chemical manipulation. Only by extensive dialysis against 0.5 M ZnCl2 or CoCl2 at pH 5.4 was it possible to reconstitute approximately 50% of the molecules in the Cu2Zn2 or Cu2Co2 form. The new derivatives have been characterized through EPR, CD and nuclear magnetic relaxation dispersion techniques. The Cu2Cox derivatives (x approximately 1) were used to monitor, through electronic and 1H-NMR spectroscopies, the metal sites which are found to be similar to those of the wild type. In addition, a double substitution with asparagine has been made, replacing the invariant aspartate at position 124 and the highly conserved aspartate at position 125. The behavior is similar to that of the other mutants in most respects. The Cu2E2 (E = empty) derivatives of the mutants are stable, even in the pH range 8-10, whereas in the case of the Cu2E2 derivative of the wild type, copper migration occurs at high pH, producing both Cu2Cu2 and apo derivatives. The activity measurements indicate that the various Cu2E2 derivatives have the same activity at low pH and similar to that of the holoenzyme. A full profile up to pH 10.5 was obtained for the mutants.
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PMID:A characterization of copper/zinc superoxide dismutase mutants at position 124. Zinc-deficient proteins. 184 81

600-MHz 1H-NMR and nuclear Overhauser enhancement spectroscopy (NOESY) spectra in 2H2O and H2O, as well as truncated driven NOE difference spectra in H2O of reduced human Cu(II)2Zn(II)2 superoxide dismutase (Cu/Zn SOD) have been recorded and used to assign the active-site proton signals. A derivative with histidines selectively deuteriated in the C2 position has been used for the detection of the HC2 histidine protons, 16 out of 17 observed signals of the 18 active-site histidine ring protons have been assigned. The results are compared with previous proposals based on more limited data sets. The numerous cross peaks confirm that the structure in solution is essentially similar to the crystallographic data obtained on the oxidized form. Probably this holds also for His63 which in the reduced form is not bridging any more the two metal ions. The effects of azide binding on the exchangeable 1H-NMR signals of the reduced protein are also reported.
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PMID:Assignment of active-site protons in the 1H-NMR spectrum of reduced human Cu/Zn superoxide dismutase. 185 82

113Cd nuclear magnetic resonance spectroscopy has been used to investigate the metal binding sites of cadmium-substituted copper, zinc-containing superoxide dismutase from baker's yeast. NMR signals were obtained for 113Cd(II) at the Cu site as well as for 113Cd(II) at the Zn site. The two subunits in the dimeric enzyme were found to have identical coordination properties towards 113Cd(II) at the Zn site when no copper is coordinated at the Cu site, and when Cu(I) or Cd(II) is coordinated, were found to be very small indicating that 113Cd(II) must be bound to the same number and type of ligands in both cases. Furthermore, the spectra show that the rate of exchange of protein-bound 113Cd(II) and free 113Cd2+ is slow on the NMR time scale also at the Cu site. The present study suggests an explanation for the discrepancy in the literature regarding 113Cd-NMR investigations of bovine superoxide dismutase.
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PMID:113Cd-NMR investigation of a cadmium-substituted copper, zinc-containing superoxide dismutase from yeast. 205 Jan 41

Thiourea and superoxide dismutase were effective antidotes to paraquat toxicity in an HL60 cell culture system, whereas other hydroxyl scavengers were ineffective. The efficacy of thioureas was not due to blockage of intracellular paraquat uptake, inhibition of NADPH-P-450 reductase, or reaction with the paraquat radical. Thiourea also competitively inhibited the reduction of cytochrome c by the xanthine/xanthine oxidase superoxide-generating system, and the release of iron from ferritin by superoxide radicals. The reaction of superoxide with thiourea produced a sulfhydryl compound distinct from products formed by hydrogen peroxide or hydroxyl radicals. Spectrophotometric and chromatographic studies indicated the carbon-sulfide double bond was converted to a sulfhydryl group which reacted with Ellman's reagent. Additional confirmatory evidence for the sulfhydryl compound was obtained with carbon-13 NMR and mass spectroscopies. Thus, thioureas are direct scavengers of superoxide radicals as well as hydroxyl radicals and hydrogen peroxide. The rate constant for the reduction of thiourea by superoxide was estimated at 1.1 x 10(3) M-1 s-1. The implication of this finding on free radical studies, the mechanism of paraquat toxicity, and the metabolism of thioureas is discussed.
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PMID:Thioureas react with superoxide radicals to yield a sulfhydryl compound. Explanation for protective effect against paraquat. 215 25


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