共查询到20条相似文献,搜索用时 31 毫秒
1.
Gogliettino MA Tanfani F Sciré A Ursby T Adinolfi BS Cacciamani T De Vendittis E 《Biochemistry》2004,43(8):2199-2208
We have examined and compared the effects of mutating Y41 and H155 in the iron superoxide dismutase (SOD) from the archaeon Sulfolobus solfataricus (Ss). These two neighboring residues in the active site are known to have crucial functions in structurally related SODs from different sources. The metal analysis indicates a slightly lower iron content after either Y41F or H155Q replacement, without any significant substitution of iron for manganese. The specific activity of SsSOD referred to the iron content is 17-fold reduced in the Y41F mutant, whereas it is less than 2-fold reduced by the H155Q mutation. The noticeable pH dependence of the activity of SsSOD and H155Q-SsSOD, due to the ionization of Y41 (pK 8.4), is lost in Y41F-SsSOD. After H155Q and even more after the Y41F substitution, the archaeal enzyme acquires a moderate sensitivity to sodium azide inhibition. The hydrogen peroxide inactivation of SsSOD is significantly increased after H155Q replacement; however, both mutants are insensitive to the modification of residue 41 by phenylmethanesulfonyl fluoride. Heat inactivation studies showed that the high stability of SsSOD is reduced by the H155Q mutation; however, upon the addition of SDS, a much faster inactivation kinetics is observed both with wild-type and mutant SsSOD forms. The detergent is also required to follow thermal denaturation of the archaeal enzyme by Fourier transform infrared spectroscopy; these studies gave information about the effect of mutations and modification on flexibility and compactness of the protein structure. The crystal structure of Y41F mutant revealed an uninterrupted hydrogen bond network including three solvent molecules connecting the iron-ligating hydroxide ion via H155 with F41 and H37, which is not present in structures of the corresponding mutant SODs from other sources. These data suggest that Y41 and H155 are important for the structural and functional properties of SsSOD; in particular, Y41 seems to be a powerful regulator of the activity of SsSOD, whereas H155 is apparently involved in the organization of the active site of the enzyme. 相似文献
2.
Specific cleavage of human type III collagen by human polymorphonuclear leukocyte elastase 总被引:11,自引:0,他引:11
C L Mainardi D L Hasty J M Seyer A H Kang 《The Journal of biological chemistry》1980,255(24):12006-12010
Purified polymorphonuclear leukocyte elastase degraded native human liver type III collagen at 27 degrees C by making a cleavage through the triple helix. The enzyme had no effect on human type I collagen. The reaction was inhibited by phenylmethanesulfonyl fluoride (PhCH2SO2F) but not by EDTA. The collagen reaction products were identical with those generated by human rheumatoid synovial collagenase when analyzed by polyacrylamide gel electrophoresis and gel filtration. NH2-trminal sequence analysis indicated that the enzyme cleaved at an isoleucyl-threonyl bond located 4 residues on the carboxyl side of the established cleavage site for animal collagenases. Therefore, it is likely that in pathologic states, type III collagen can be selectively depleted from the matrix by this enzyme. 相似文献
3.
Phosphate was reported to be an inhibitor of copper- and zinc-containing superoxide dismutase (SOD) [de Freitas, D.M., & Valentine, J.S. (1984) Biochemistry 23, 2079-2082]. Thus SOD activity, in 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.4), was decreased by approximately 50% when the assay was made 10 mM in phosphate, and the ionic strength was adjusted with sodium fluoride. The inhibitory effect of phosphate was attributed to the neutralization of the positive charge on the guanidino residue of Arg-141. We have reexamined the effects of phosphate inhibition of SOD and found that the enzyme has identical activity in phosphate or HEPES buffer when the ionic strength is adjusted with NaBr. The putative inhibitory effect of phosphate appears to have been due to fluoride inhibition of the superoxide generating system of xanthine/xanthine oxidase. We have confirmed this result by using a photochemical generation of O2- in addition to the enzymatic generation of O2-. Chemical modification of the lysine residues to homoarginines does not affect the activity of the enzyme and does not impart a phosphate sensitivity. Chemical modification with phenylglyoxal caused approximately 80% inactivation of the native enzyme and 90% inactivation of the O-methylisourea-modified enzyme. Our results suggest that phosphate does not inhibit the copper- and zinc-containing superoxide dismutase (Cu,Zn-SOD) beyond the expectations of its effect on ionic strength. 相似文献
4.
Helena Danielsson Thorell Natascha H Beyer Niels H H Heegaard Marcus Ohman Thomas Nilsson 《European journal of biochemistry》2004,271(17):3539-3546
A detailed comparison between native chlorite dismutase from Ideonella dechloratans, and the recombinant version of the protein produced in Escherichia coli, suggests the presence of a covalent modification in the native enzyme. Although the native and recombinant N- and C-terminal sequences are identical, the enzymes display different electrophoretic mobilities, and produce different peptide maps upon digestion with trypsin and separation of fragments using capillary electrophoresis. Comparison of MALDI mass spectra of tryptic peptides from the native and recombinant enzymes suggests two locations for modification in the native protein. Mass spectrometric analysis of isolated peptides from a tryptic digest of the native enzyme identifies a possible cross-linked dipeptide, suggesting an intrachain cross-link in the parent protein. Spectrophotometric titration of the native enzyme in the denatured state reveals two titrating components absorbing at 295 nm, suggesting the presence of about one tyrosine residue per subunit with an anomalously low pK(a). The EPR spectrum for the recombinant enzyme is different from that of the native enzyme, and contains a substantial contribution of a low-spin species with the characteristics of bis-histidine coordination. These results are discussed in terms of a covalent cross-link between a histidine and a tyrosine sidechain, similar to those found in other heme enzymes operating under highly oxidizing conditions. 相似文献
5.
L Banci I Bertini P Caliceti L Monsù Scolaro O Schiavon F M Veronese 《Journal of inorganic biochemistry》1990,39(2):149-159
Spectroscopic methods have been employed in order to understand the molecular basis of the decrease in enzymatic activity of the antiinflammatory enzyme copper-zinc superoxide dismutase (SOD) following the covalent binding of polyethyleneglycol (PEG) chains to the protein amino-groups. The PEG modification is a general method recently proposed to improve the therapeutic index of enzymes. 1H NMR spectra on the cobalt substituted PEG-modified SOD, Cu2Co2-PEG-SOD, have been recorded. The signals are quite broad with respect to the unmodified enzyme. This has been interpreted on the basis of the effect of molecular weight on the linewidth. The analysis has shown that the histidine hydrogens involved in metal binding at the enzyme active site are the same in both native and PEG-modified SOD. Similarly, circular dichroism and absorption spectra indicate that the overall conformation of the metal clusters is not perturbed upon modification. On the other hand, azide titration shows that the affinity constant of N-3 for SOD is largely reduced upon PEG modification (K = 154 M-1 and 75 M-1 for the native and modified SOD, respectively). These results indicate that the decrease in enzymatic activity upon surface modification with PEG is not caused by a perturbation of the active site geometry, but to a decrease in the channeling of the O2- ion towards the enzyme active site. 相似文献
6.
A method was worked out that helped us to isolate superoxide dismutase (SOD) from human blood plasma. The change of enzyme's activity was shown depending on the period of SOD storage. Changes in activity were observed in storing protein after gel filtration. The activity of purified enzyme was half as much after 24 h storage and remained constant for a long period of time (5 mo). The change of SOD activity was found to be connected with a modification of its structure. The storage of enzyme's solution during 3 1/2 mo is accompanied by the lowering of protein molecular mass from 53,000 Da to 34,000 Da. The inhibitors of proteinases--phenyl-methylsulfonyl fluoride (PMSF) and alpha 2-macroglobulin--showed no protective effects on purified SOD. That's why it was possible to say that the lowering of protein molecular mass didn't connect with a specific proteolysis. An oxidative modification of SOD structure is under discussion now. The modification is most probably caused by oxidative destruction of aminoacid residues that are located outside the protein active centre. 相似文献
7.
Contribution of a tyrosine side chain to ribonuclease A catalysis and stability. 总被引:1,自引:1,他引:0
下载免费PDF全文
![点击此处可从《Protein science : a publication of the Protein Society》网站下载免费的PDF全文](/ch/ext_images/free.gif)
E. S. Eberhardt P. K. Wittmayer B. M. Templer R. T. Raines 《Protein science : a publication of the Protein Society》1996,5(8):1697-1703
An intricate architecture of covalent bonds and noncovalent interactions appear to position the side chain of Lys 41 properly within the active site of bovine pancreatic ribonuclease A (RNase A). One of these interactions arises from Tyr 97, which is conserved in all 41 RNase A homologues of known sequence. Tyr 97 has a solvent-inaccessible side chain that donates a hydrogen bond to the main-chain oxygen of Lys 41. Here, the role of Tyr 97 was examined by replacing Tyr 97 with a phenylalanine, alanine, or glycine residue. All three mutant proteins have diminished catalytic activity, with the value of Kcat being perturbed more significantly than that of Km. The free energies with which Y97F, Y97A, and Y97G RNase A bind to the rate-limiting transition state during the cleavage of poly(cytidylic acid) are diminished by 0.74, 3.3, and 3.8 kcal/mol, respectively. These results show that even though Tyr 97 is remote from the active site, its side chain contributes to catalysis. The role of Tyr 97 in the thermal stability of RNase A is large. The conformational free energies of native Y97F, Y97A, and Y97G RNase A are decreased by 3.54, 12.0, and 11.7 kcal/mol, respectively. The unusually large decrease in stability caused by the Tyr-->Phe mutation could result from a decrease in the barrier to isomerization of the Lys 41-Pro 42 peptide bond. 相似文献
8.
D E Cabelli Y Guan V Leveque A S Hearn J A Tainer H S Nick D N Silverman 《Biochemistry》1999,38(36):11686-11692
Tryptophan 161 is a highly conserved residue that forms a hydrophobic side of the active site cavity of manganese superoxide dismutase (MnSOD), with its indole ring adjacent to and about 5 A from the manganese. We have made a mutant containing the conservative replacement Trp 161 --> Phe in human MnSOD (W161F MnSOD), determined its crystal structure, and measured the catalysis of the resulting mutant using pulse radiolysis to produce O(2)(*)(-). In the structure of W161F MnSOD the phenyl side chain of Phe 161 superimposes on the indole ring of Trp 161 in the wild type. However, in the mutant, the hydroxyl side chain of Tyr 34 is 3.9 A from the manganese, closer by 1.2 A than in the wild type. The tryptophan in MnSOD is not essential for the half-cycle of catalytic activity involving reduction of the manganese; the mutant W161F MnSOD had k(cat)/K(m) at 2.5 x 10(8) M(-)(1) s(-)(1), reduced only 3-fold compared with wild type. However, this mutant exhibited a strong product inhibition with a zero-order region of superoxide decay slower by 10-fold compared with wild type. The visible absorption spectrum of W161F MnSOD in the inhibited state was very similar to that observed for the inhibited wild-type enzyme. The appearance of the inhibited form required reaction of 2 molar equiv of O(2)(*)(-) with W161F Mn(III)SOD, one to form the reduced state of the metal and the second to form the inhibited complex, confirming that the inhibited complex requires reaction of O(2)(*)(-) with the reduced form of the enzyme. This work suggests that a significant role of Trp 161 in the active site is to promote the dissociation of product peroxide, perhaps in part through its effect on the orientation of Tyr 34. 相似文献
9.
The ground state magnetic properties of manganese superoxide dismutase from Thermus thermophilus in its native and reduced forms have been determined using saturation magnetization data. Parallel EPR measurements were used to verify that commonly encountered paramagnetic impurities were at low concentration relative to the metalloprotein. The native enzyme contains high spin Mn(III) (S = 2) with D = +2.44(5) cm-1 and E/D = 0. The reduced enzyme contains high spin Mn(II) (S = 5/2) with D = +0.50(5) cm-1 and E/D = 0.027. These results are in keeping with the suggestions of several previous groups of workers concerning the permissible oxidation and spin states of the manganese, but the zero field splitting parameters are unlike those of known manganese model compounds. In addition, the extinction coefficient for the visible region absorption maximum of the native enzyme and the corresponding difference extinction coefficient (native minus reduced) have been measured using saturation magnetization data to quantitate Mn(III) present. The result, epsilon 480 = 950(80) M-1 cm-1 (delta epsilon 480 = 740(60) M-1 cm-1) agrees with the previously reported value of epsilon 480 = 910 M-1 cm-1 found by total manganese determination (Sato, S. and Nakazawa, K. (1978) J. Biochem. 83, 1165-1171). The wide variation in the reported visible region extinction coefficients of manganese superoxide dismutases from different sources is discussed. 相似文献
10.
Wang Z He Z Shen Q Gu Y Li S Yuan Q 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2005,826(1-2):114-121
The cDNA of Cu, Zn containing superoxide dismutase from the Cordyceps militaris SH (cm-SOD) was overexpressed in Escherichia coli BL 21 (DE3) using the pET-21a expression vector. The recombinant cell overexpressed the protein corresponding to 35+/-3% of total bacterial protein in cytosol. The purification was performed through three steps: DEAE-FF, CM-52, and G-100. After this purification procedure, a specific activity of 27272.7 U/mg of protein was reached, corresponding to 6.1-fold purification with a yield of 85.0%. The purity was homogeneous by SDS-PAGE analysis and 94.2+/-1.0% by CZE analysis. A subunit molecular mass of the recombinant enzyme was 15704 Da with a Cu and Zn element. In addition, the dimeric and polymeric structures were observed on MALDI-TOF-MS. Isoelectric point value of 7.0 was obtained for the recombinant enzyme that was sensitive to H2O2 and KCN. The recombinant enzyme remained 80+/-2% residual activity at pH 7.8, at 50 degrees C for 4h incubation. The properties: N-terminal amino acid sequence (the first 12 amino acid residues), pI, subunit molecular mass, thermo-stability of the purified recombinant SOD are similar to that of the native Cu, Zn-SOD from C. militaris (N-cm-SOD). 相似文献
11.
Quint P Ayala I Busby SA Chalmers MJ Griffin PR Rocca J Nick HS Silverman DN 《Biochemistry》2006,45(27):8209-8215
Human manganese superoxide dismutase (MnSOD) is a homotetramer of 22 kDa subunits, a dimer of dimers containing dimeric and tetrameric interfaces. We have investigated conformational mobility at these interfaces by measuring amide hydrogen/deuterium (H/D) exchange kinetics and 19F NMR spectra, both being excellent methods for analyzing local environments. Human MnSOD was prepared in which all nine tyrosine residues in each subunit are replaced with 3-fluorotyrosine. The 19F NMR spectrum of this enzyme showed five sharp resonances that have been assigned by site-specific mutagenesis by replacing each 3-fluorotyrosine with phenylalanine; four 19F resonances not observed are near the paramagnetic manganese and extensively broadened. The temperature dependence of the line widths and chemical shifts of the 19F resonances were used to estimate conformational mobility. 3-Fluorotyrosine 169 at the dimeric interface showed little conformational mobility and 3-fluorotyrosine 45 at the tetrameric interface showed much greater mobility by these measures. In complementary studies, H/D exchange mass spectrometry was used to measure backbone dynamics in human MnSOD. Using this approach, amide hydrogen exchange kinetics were measured for regions comprising 78% of the MnSOD backbone. Peptides containing Tyr45 at the tetrameric interface displayed rapid exchange of hydrogen with deuterium while peptides containing Tyr169 in the dimeric interface only displayed moderate exchange. Taken together, these studies show that residues at the dimeric interface, such as Tyr169, have significantly less conformational freedom or mobility than do residues at the tetrameric interface, such as Tyr45. This is discussed in terms of the role in catalysis of residues at the dimeric interface. 相似文献
12.
Comparison of the crystal structures of genetically engineered human manganese superoxide dismutase and manganese superoxide dismutase from Thermus thermophilus: differences in dimer-dimer interaction.
下载免费PDF全文
![点击此处可从《Protein science : a publication of the Protein Society》网站下载免费的PDF全文](/ch/ext_images/free.gif)
U. G. Wagner K. A. Pattridge M. L. Ludwig W. C. Stallings M. M. Werber C. Oefner F. Frolow J. L. Sussman 《Protein science : a publication of the Protein Society》1993,2(5):814-825
The three-dimensional X-ray structure of a recombinant human mitochondrial manganese superoxide dismutase (MnSOD) (chain length 198 residues) was determined by the method of molecular replacement using the related structure of MnSOD from Thermus thermophilus as a search model. This tetrameric human MnSOD crystallizes in space group P2(1)2(1)2 with a dimer in the asymmetric unit (Wagner, U.G., Werber, M.M., Beck, Y., Hartman, J.R., Frolow, F., & Sussman, J.L., 1989, J. Mol. Biol. 206, 787-788). Refinement of the protein structure (3,148 atoms with Mn and no solvents), with restraints maintaining noncrystallographic symmetry, converged at an R-factor of 0.207 using all data from 8.0 to 3.2 A resolution and group thermal parameters. The monomer-monomer interactions typical of bacterial Fe- and Mn-containing SODs are retained in the human enzyme, but the dimer-dimer interactions that form the tetramer are very different from those found in the structure of MnSOD from T. thermophilus. In human MnSOD one of the dimers is rotated by 84 degrees relative to its equivalent in the thermophile enzyme. As a result the monomers are arranged in an approximately tetrahedral array, the dimer-dimer packing is more intimate than observed in the bacterial MnSOD from T. thermophilus, and the dimers interdigitate. The metal-ligand interactions, determined by refinement and verified by computation of omit maps, are identical to those observed in T. thermophilus MnSOD. 相似文献
13.
Greenleaf WB Perry JJ Hearn AS Cabelli DE Lepock JR Stroupe ME Tainer JA Nick HS Silverman DN 《Biochemistry》2004,43(22):7038-7045
The side chain of Gln143, a conserved residue in manganese superoxide dismutase (MnSOD), forms a hydrogen bond with the manganese-bound solvent and is critical in maintaining catalytic activity. The side chains of Tyr34 and Trp123 form hydrogen bonds with the carboxamide of Gln143. We have replaced Tyr34 and Trp123 with Phe in single and double mutants of human MnSOD and measured their catalytic activity by stopped-flow spectrophotometry and pulse radiolysis. The replacements of these side chains inhibited steps in the catalysis as much as 50-fold; in addition, they altered the gating between catalysis and formation of a peroxide complex to yield a more product-inhibited enzyme. The replacement of both Tyr34 and Trp123 in a double mutant showed that these two residues interact cooperatively in maintaining catalytic activity. The crystal structure of Y34F/W123F human MnSOD at 1.95 A resolution suggests that this effect is not related to a conformational change in the side chain of Gln143, which does not change orientation in Y34F/W123F, but rather to more subtle electronic effects due to the loss of hydrogen bonding to the carboxamide side chain of Gln143. Wild-type MnSOD containing Trp123 and Tyr34 has approximately the same thermal stability compared with mutants containing Phe at these positions, suggesting the hydrogen bonds formed by these residues have functional rather than structural roles. 相似文献
14.
Silver-copper and silver-cobalt proteins have been prepared in which Ag+ resides in the native copper site of superoxide dismutase and either Cu2+ of Co2+ reside in the zinc site. The electron paramagnetic resonance (EPR) spectrum of the copper and the visible absorption spectrum of the cobalt greatly resemble those of either Cu4 of Cu2,Cu2,Co2 proteins, respectively, in which the copper of the native copper sites has been reduced. It was found that, unlike cyanide, azide anion would not perturb the EPR spectrum of Ag2,Cu2 protein. Since azide produces the same perturbation upon the EPR spectrum of native and Cu2 proteins, it must bind to the copper and not the zinc of superoxide dismutase. A model of the metal sites of the enzyme has been fitted to a 3-A electron-density map using an interactive molecular graphics display. The model shows that histidine-61, which appears to bind both copper and zinc, does not lie in the plane of the copper and its three other histidine ligands, but occupies a position intermediate between planar and axial. This feature probably accounts for the rhombicity of the EPR spectrum and the activity of the enzyme. 相似文献
15.
Recombinant human extracellular superoxide dismutase produced in milk of transgenic rabbits 总被引:1,自引:0,他引:1
Stromqvist Mats Houdebine Louis-Marie Andersson Jan-Olof Edlund Anders Johansson Thore Viglietta Celine Puissant Claudine Hansson Lennart 《Transgenic research》1997,6(4):271-278
Expression of human extracellular superoxide dismutase (EC-SOD), a glycosylated, tetrameric metalloprotein, was targeted to the lactating mammary gland of transgenic rabbits. Efficient expression of the recombinant whey acidic protein/ec-sod gene was achieved and up to 3 mg ml–1 of the enzyme was secreted into the milk. Rabbit milk-produced recombinant EC-SOD was primarily found in the whey and purified by a two-step chromatographic method. To evaluate the rabbit milk-produced human EC-SOD, comparisons with native and Chinese hamster ovary cell (CHO)-produced EC-SOD were performed. All proteins were tetrameric and N-glycosylated. The behaviour on SDS-PAGE and size-exclusion chromatography indicated that the masses, and thereby the extent of post-translational modification of the proteins was similar. The monosaccharide composition of both recombinant EC-SOD variants was analysed and indicated similarities in the attached N-glycans on the two proteins. Furthermore, the peptide maps of the three EC-SOD variants revealed that all proteins had similar polypeptide backbones 相似文献
16.
Biological protection by superoxide dismutase 总被引:10,自引:0,他引:10
F Lavelle A M Michelson L Dimitrijevic 《Biochemical and biophysical research communications》1973,55(2):350-357
Diol dehydrase from was dissociated into two different protein components or subunits, designated Components F and S, by chromatography on DEAE-cellulose. Neither component alone possessed any appreciable catalytic activity. Diol dehydrase activity was restored when the two components were combined. Both components were also required for inactivation of coenzyme B12 by oxygen when incubation was carried out in the absence of substrate aerobically. The more acidic component, Component S, was a sulfhydryl protein sensitive to an alkylating agent, iodoacetamide. Coenzyme B12 was not bound by the individual components, F or S, both of which were necessary for the cobamide binding. The presence of substrate, 1,2-propanediol, in eluting buffer retarded the dissociation of the enzyme. 相似文献
17.
Castellano I Ruocco MR Cecere F Di Maro A Chambery A Michniewicz A Parlato G Masullo M De Vendittis E 《Biochimica et biophysica acta》2008,1784(5):816-826
Our previous work showed that the adduct between beta-mercaptoethanol and the single cysteine residue (Cys57) in superoxide dismutase from the psychrophilic eubacterium Pseudoalteromonas haloplanktis (PhSOD) reduces the enzyme inactivation by peroxynitrite. In this work, immunoblotting experiments prove that peroxynitrite inactivation of PhSOD involves formation of nitrotyrosine residue(s). In order to study the role of Cys57 as a redox-sensor residue modifiable by cellular thiols, a recombinant PhSOD and two Cys57 mutants were produced and characterized. Recombinant and mutant enzymes share similar activity and peroxynitrite inactivation, but different reactivity towards three glutathione forms. Indeed, oxidized glutathione and S-nitrosoglutathione, but reduced glutathione, lead to S-glutathionylation of recombinant PhSOD. This new covalent modification for a Fe-SOD does not occur in both Cys57 mutants, thus indicating that its target is Cys57. Moreover, mass spectrometry analysis confirmed that S-glutathionylation of Cys57 takes place also with endogenous PhSOD. Formation of this mixed disulfide in PhSOD protects the enzyme from tyrosine nitration and peroxynitrite inactivation. PhSOD undergoes S-glutathionylation during its overproduction in E. coli cells and in a growing culture of P. haloplanktis. In both cases the extent of glutathionylated PhSOD is enhanced upon cell exposure to oxidative agents. We suggest that S-glutathionylation of PhSOD could represent a further cold-adaptation strategy to improve the antioxidant cellular defence mechanism. 相似文献
18.
Fumiyuki Yamakura Hiroaki Kawasaki 《Biochimica et Biophysica Acta - Proteins and Proteomics》2010,1804(2):318-325
Post-translational modifications of proteins control many biological processes through the activation, inactivation, or gain-of-function of the proteins. Recent developments in mass spectrometry have enabled detailed structural analyses of covalent modifications of proteins and also have shed light on the post-translational modification of superoxide dismutase. In this review, we introduce some covalent modifications of superoxide dismutase, nitration, phosphorylation, glutathionylaion, and glycation. Nitration has been the most extensively analyzed modification both in vitro and in vivo. Reaction of human Cu,Zn superoxide dismutase (SOD) with reactive nitrogen species resulted in nitration of a single tryptophan residue to 6-nitrotryptophan, which could be a new biomarker of a formation of reactive nitrogen species. On the other hand, tyrosine 34 of human MnSOD was exclusively nitrated to 3-nitrotyrosine and almost completely inactivated by the reaction with peroxynitrite. The nitrated MnSOD has been found in many diseases caused by ischemia/reperfusion, inflammation, and others and may have a pivotal role in the pathology of the diseases. Most of the post-translational modifications have given rise to a reduced activity of SOD. Since phosphorylation and nitration of SOD have been shown to have a possible reversible process, these modifications may be related to a redox signaling process in cells. Finally we briefly introduce a metal insertion system of SOD, focusing particularly on the iron misincorporation of nSOD, as a part of post-translational modifications. 相似文献
19.
A pulse-radiolysis study of the manganese-containing superoxide dismutase from Bacillus stearothermophilus.
下载免费PDF全文
![点击此处可从《The Biochemical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
In the preceding paper the mechanism of catalysis of the manganese-containing superoxide dismutase from Bacillus stearothermophilus was shown to involve a 'fast cycle' and a 'slow cycle' [McAdam, Fox, Lavelle & Fielden, 1977 (Biochem. J. 165, 71-79)]. Further properties of the enzyme was considered in the present paper. Pulse-radiolysis studies, under conditions of low substrate concentration to (i.e. when the fast cycle predominates), showed that enzyme activity decreases as pH increases (6.5-10.2). Activity was unaffected by the addition of H2O2 or NaN3 but slightly decreased by KCN. Both H2O2 and the reducing radical anion CO2-- caused a decrease in A480 of the native enzyme. The rate of the fast catalytic cycle was independent of temperature (5-55 degrees C), and as temperature increases the slow cycle becomes relatively more important. Arrhenius parameters of the rate contants were estimated. The possible identity of the various forms of the enzyme is considered. 相似文献
20.
The redox potential of human manganese superoxide dismutase (MnSOD) has been difficult to determine because of the problem of finding suitable electron mediators. We have found that ferricyanide and pentacyanoaminoferrate can be used as electron mediators, although equilibration is very slow with a half-time near 6 h. Values of the midpoint potential were determined both by allowing enzyme and mediators to equilibrate up to 38 h and by reductive titration adding dithionite to enzyme and mediator. An overall value of the midpoint potential was found to be 393 +/- 29 mV. To elucidate the role of His30 and Tyr34 in the active site of human MnSOD, we have also measured the redox properties of the site-specific mutants His30Asn (H30N) and Tyr34Phe (Y34F) and compared them with the wild-type enzyme. Crystal structures have shown that each mutation interrupts a hydrogen bond network in the active site, and each causes a 10-fold decrease in the maximal velocity of catalysis of superoxide dismutation as compared with wild type. The present study shows that H30N and Y34F human MnSOD have very little effect, within experimental uncertainty, on the redox potential of the active-site metal. The redox potentials determined electrochemically were 365 +/- 28 mV for H30N and 435 +/- 30 mV for Y34F MnSOD. These results suggest that the role of His30 and Tyr34 is more in support of catalysis, probably proton transport, and not in the tuning of the redox potential. 相似文献