Phosphate is an inhibitor of copper-zinc superoxide dismutase

Freitas, Duarte Mota de; Valentine, Joan Selverstone

doi:10.1021/bi00304a031

Cited by 52 publications

(35 citation statements)

References 23 publications

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“…The dissociation constant for the azide binding (6mM) is about two times smaller than that of the azide binding to superoxide dismutase (Mota de Freitas & Valentine, 1984). This is in line with the stabilizing effect of the residues surrounding azide in the Met l21Ala mutant.…”

Section: The Azjde Derivative Of the Metl21ala Mutantsupporting

confidence: 60%

Mutant Met121Ala ofPseudomonas aeruginosaAzurin and Its Azide Derivative: Crystal Structures and Spectral Properties

Tsai¹,

Bonander²,

Harata³

et al. 1996

Acta Cryst D

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The crystal structures of the azurin mutant Met l21Ala and its azide derivative Met l21Ala-azide from Pseudomonas aeruginosa have been determined. The final crystallographic R values are 21.3 and 19.4% for the two structures, respectively. In the Met l21Ala mutant, the distance between the copper ion and His117 increases by 0.34/~ compared with the wild-type structure. The removal of the methionine in the apical position induces a shortening of the distance from the copper ion to the carbonyl O atom of Gly45 from 2.97 to 2.74/~. In the Met l 21Ala-azide structure, the azide anion occupies the cavity created by replacing the Met l21 side chain with the smaller methyl group of Ala. The azide anion binds with a terminal N atom to the copper ion at a distance of about 2.04 A. In addition, the copper ion has moved out of the trigonal plane by about 0.26 A, towards the azide anion. Thus, the copper site in this structure has a distorted tetrahedral arrangement. The spectroscopic characteristics show, in addition, that the copper sites in the two structures are distinctively different. The Met l21Ala mutant still maintains the properties of an ordinary type 1 copper site while the Metl21Ala-azide derivative has an absorption maximum at about 409 nm and the copper hyperfine coupling has increased to a value intermediate between those of type 2 copper and the wild-type azurin.

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Section: The Azjde Derivative Of the Metl21ala Mutantsupporting

confidence: 60%

Mutant Met121Ala ofPseudomonas aeruginosaAzurin and Its Azide Derivative: Crystal Structures and Spectral Properties

Tsai¹,

Bonander²,

Harata³

et al. 1996

Acta Cryst D

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“…either the active site copper or Arg-141 [ 17]. The results indicate that an anionic scavenger, azide, has easy access inside the positively active channel of both enzymes and can compete with ABTS for the .OH radicals.…”

Section: Resultsmentioning

confidence: 90%

The free radical‐generating function of a familial amyotrophic lateral sclerosis‐associated D90A Cu,Zn‐superoxide dismutase mutant

Kim¹,

Eum²,

Kwon³

et al. 1998

IUBMB Life

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SUMMARYThe free radical-generating functions of the D90A Cu,Zn-superoxide dismutase (SOD) associated with Swedish familial amyotrophic lateral sclerosis (FALS) patients are investigated. The results show that both the wild-type and mutant enzymes have identical dismutase activity, while the free radical-generating activity of the D90A mutant is enhanced relative to that of the wild-type enzyme. The studies suggest that the active channel of the D90A mutant is larger than that of the wild-type enzyme. A higher free radical-generating activity of the mutant enzyme led to the release of copper ions from the damaged protein. The generation of strand breaks in plasmid DNA was enhanced more effectively by the D90A mutant Cu,Zn-SOD than by the wildtype enzyme. The results suggest that the pathology of FALS may be attributed to oxidative damage caused by the gain-of-function of FALS Cu,Zn-SOD mutant.Key words: copper,zin-superoxide dismutase, mutant, hydroxyl radical, copper. 1191All rights of reproduction in any form reserved, Vol. 46, No. 6, 1998 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONALAndersen et al. [7] reported a Cu,Zn-SOD exon 4 mutation (D90A) with novel features in Swedish FALS patients and the disease has only been observed in patients homozygous for this mutation, whereas heterozygotes remained unaffected. Furthermore, there was essentially no reduction in erythrocyte Cu,Zn-SOD dismutation activity. Thus, [7] suggested that this Cu,Zn-SOD mutation caused ALS by a gain-of-function rather than by the loss of dismutation activity.In view of these considerations, the present work represents an investigation of the free radical-generating functions of the D90A mutant and the oxidative damage to DNA, as a first attempt to establish the extent of any correlation between the gain-of-function and the pathology of FALS. MATERIALS AND METHODS Expression and purification of wild-type and mutated human Cu, Zn-SOD

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“…In addition to the usual superoxide dismutation activity, Cu,Zn-SOD is known to exhibit anion binding capacity (9,10), inactivation by its own reaction product H202 (11)(12)(13), and the purported peroxidase activity (14). Here we summarize our findings that Cu,Zn-SOD has free radical ('OH and scavenger-derived radicals)-generating activity (15,16).…”

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confidence: 78%

Pro-oxidant activity of Cu,Zn-superoxide dismutase

Yim

Chock

et al. 1998

AGE

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Whereas Cu,Zn-superoxide dismutase (Cu,Zn-SOD) is an essential antioxidant enzyme for superoxide dismutation in vivo(1 ), many reports have shown that an elevated level of Cu,Zn-SOD induces cell killing (2, 3), increases lipid peroxidation (4,5), and interferes with the transport of neurotransmitters (4, 6). All of these observations are archetypical results inducible by oxygen radicals. Cu,Zn-SOD (7) and FALS mutants (8) are known to develop neurodegeneration. These findings suggest that under certain conditions Cu,Zn-SOD may behave directly or indirectly as a pro-oxidant.In addition to the usual superoxide dismutation activity, Cu,Zn-SOD is known to exhibit anion binding capacity (9, 10), inactivation by its own reaction product H202 (11-13), and the purported peroxidase activity (14). Here we summarize our findings that Cu,Zn-SOD has free radical ('OH and scavenger-derived radicals)-generating activity (15,16). TheactiveCu,Zn-SOD can catalyze the generation of free 9 radicals from H202. In the presence of H202 and radical scavengers, especially with anionic character, both free 9 radicals and scavenger-derived radicals are produced (15, 16). We also found that the free radical-generating function of several FALS-associated Cu,Zn-SOD mutants is enhanced relative to that of the wild-type enzyme, while the dismutation activities remain unchanged (17,18).To monitor the production of free radicals, two spin traps, 5,5-dimethyl-l-pyrroline N-oxide (DMPO) and Ntert-butyl-c~-phenylnitrone (PBN), were used in our studies (15) to convert transient free radicals to stable free radicals adducts according to the following reactions:The nature of the trapped free radical was identified by EPR spectroscopy. When O2-" (dissolved KO 2 in dried dimethylsulfoxide) was added to the NaHCOJCO 2 buffer at pH 7.6 containing 100 mM DMPO, EPR OH adducts. DMPO-OH formation also required active Cu,Zn-SOD, because heat-inactivated Cu,Zn-SOD or Mn-SOD failed to produce these radical adducts. The direct addition of H202 in place of O2-" produced similar results. In the presence of .OH radical scavengers with anionic character such as HCO 2 or N3-, DMPO adducts of the scavenger-derived radicals, DMPO-CO2-" or DMPO-N3", were observed in addition to DMPO-OH. The concentration ratio between DMPO-OH and DMPO-scavenger-derived radical adducts observed in these experiments reflects competition reactions between DMPO and scavengers for free "OH radicals. Surprisingly, with neutral'OH radical scavengers, such as ethanol, the formation of DMPO-hydroxyethyl radical adducts was insignificant and the concentration of DMPO-OH formed was unaffected. In contrast, with a different spin trap, PBN, in place of DMPO, ethanol yields PBNhydroxyethyl radical adducts as expected in the competition reaction for free "OH radicals. These seemingly contradicting results may be caused by the different affinities of these spin traps for the positively charged active channel leading to the active site of Cu,Zn-SOD, where the'OH radicals are generated. To prove this hypo...

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Phosphate is an inhibitor of copper-zinc superoxide dismutase

Cited by 52 publications

References 23 publications

Mutant Met121Ala ofPseudomonas aeruginosaAzurin and Its Azide Derivative: Crystal Structures and Spectral Properties

Mutant Met121Ala ofPseudomonas aeruginosaAzurin and Its Azide Derivative: Crystal Structures and Spectral Properties

The free radical‐generating function of a familial amyotrophic lateral sclerosis‐associated D90A Cu,Zn‐superoxide dismutase mutant

Pro-oxidant activity of Cu,Zn-superoxide dismutase

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