Binding sites of anions in superoxide dismutase

Bertini, Ivano; Borghi, E.; Luchinat, Claudio; Scozzafava, A.

doi:10.1021/ja00416a015

Cited by 45 publications

(13 citation statements)

References 4 publications

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“…For these reasons, we chose to study the effects of phosphate on the properties of the protein with sodium fluoride to adjust ionic strength. In spite of the fact that fluoride binds weakly to copper(I1) in the protein ( E g o et al, 1977;Bertini et al, 1981;Viglino et al, 1981), fluoride seemed to maximize the effect of phosphate on the properties of the protein while giving the required concn of SOD to give 50% inhibition (ng/mL)b highest specific activity of those salts investigated (other than trifluorornethanesulfonate). As buffer for our studies, we chose Hepes because of its appropriate buffering range, its lack of affinity for metal ions, and its steric bulk, which made interference with anion binding sites on the protein unlikely.…”

Section: Resultsmentioning

confidence: 99%

Phosphate is an inhibitor of copper-zinc superoxide dismutase

Freitas¹,

Valentine²

1984

Biochemistry

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The superoxide dismutase (SOD) activity of bovine copper-zinc superoxide dismutase (Cu,Zn-SOD) in 50 mM Hepes [4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid], pH 7.4, was decreased by approximately 50% when the solution was made 10 mM in phosphate, in spite of the fact that the ionic strength of both solutions was adjusted to be equal. A similar experiment was carried out with bovine Cu,Zn-SOD chemically modified at Arg-141 with phenylglyoxal, which consequently had approximately 20% of the activity of the unmodified protein. (This activity was shown not to be due to residual unmodified protein.) Addition of 10 mM phosphate to solutions of the modified protein caused only a small decrease (less than 5%) in the SOD activity. The presence of phosphate also caused the affinity of Cu,Zn-SOD for binding azide or cyanide anions to be reduced; this effect of phosphate was also much less for the arginine-modified protein. We conclude that the inhibitory effect of phosphate on bovine Cu,Zn-SOD is due primarily to the neutralization of the positive charge on the side chain of Arg-141. The effect of increasing ionic strength on the activities of the native and arginine-modified proteins was also investigated. We found that at high concentrations of phosphate (greater than or equal to 10 mM), the SOD activities of native and arginine-modified Cu,Zn-SOD were inhibited comparably when the ionic strength was increased. This effect is presumably due to the lysine residues near the active site.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Resultsmentioning

confidence: 99%

Phosphate is an inhibitor of copper-zinc superoxide dismutase

Freitas¹,

Valentine²

1984

Biochemistry

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“…Modification of the EPR line shape, depending on the state of the protein, is a novel finding, since eukaryotic Cu,Zn SODs are known to have an identical EPR spectrum at both room and liquid nitrogen temperatures (41,42). This modification may be taken as a further indication of a slight geometrical rearrangements of the ligands surrounding the metal site in the prokaryotic as compared with that of the eukaryotic enzymes (23).…”

Section: Resultsmentioning

confidence: 99%

Cu,Zn Superoxide Dismutase from Photobacterium leiognathi Is an Hyperefficient Enzyme

Stroppolo

Sette²,

O’Neill³

et al. 1998

Biochemistry

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The catalytic rate constant of recombinant Photobacterium leiognathi Cu,Zn superoxide dismutase has been determined as a function of pH by pulse radiolysis. At pH 7 and low ionic strength (I = 0.02 M) the catalytic rate constant is 8.5 x 10(9) M-1 s-1, more than two times the value found for all the native eukaryotic Cu,Zn superoxide dismutases investigated to date. Similarly, Brownian dynamics simulations indicate an enzyme-substrate association rate more than two times higher than that found for bovine Cu,Zn superoxide dismutase. Titration of the paramagnetic contribution to the water proton relaxation rate of the P. leiognathi with increasing concentration of halide ions with different radii indicates that the proteic channel delimiting the active site is wider than 4.4 A. This is at variance with that found on the eukariotic enzymes, and provides a rationale for the high catalytic rate of the bacterial enzyme. Evidence for solvent exposure of the active site different from that observed in the eukaryotic enzyme is suggested from the pH dependence of the water proton relaxation rate and of the EPR spectrum line shape, which indicate the occurrence of a prototropic equilibrium at pH 9.1 and 9.0, respectively. The pH dependence of the P. leiognathi catalytic rate has a trend different from that observed in the bovine enzyme, indicating that groups differently exposed to the solvent are involved in the modulation of the enzyme-substrate encounter.

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“…The spectral parameters observed for component II parallel those documented above for other copper-protein-cyano complexes whose e.p.r. parameters are known to be extremely diagnostic of square-planar co-ordination (Hathaway & Billing, 1970;Bertini, 1981;Van Kamp et al, 1982). Thus component II may be assigned to a Cu(II)-cyano complex in a more planar environment than that of native dopamine mono-oxygenase.…”

Section: Discussionmentioning

confidence: 99%

Kinetic and e.p.r. studies of cyanide and azide binding to the copper sites of dopamine (3,4-dihydroxyphenethylamine) β-mono-oxygenase

Blackburn¹,

Collison²,

Sutton³

et al. 1984

Biochemical Journal

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The kinetics of inhibition of dopamine (3,4-dihydroxyphenethylamine) beta-mono-oxygenase by cyanide (CN-) and azide (N3-) ions have been investigated by using steady-state methods. Both anions show complex non-competitive-inhibition patterns with respect to ascorbate, suggestive of anion binding at two different sites on the oxidized enzyme. To further investigate this finding, e.p.r. titrations of CN- and N3- binding to the 63Cu-reconstituted enzyme were carried out. Addition of approx. 2 equiv. of CN- to copper elicits a new signal with g = 2.217, g = 2.025, A = 17.0 mT characteristic of a copper (II)-cyano complex. Simulations show that this signal accounts for half the copper (II) in the enzyme. The remainder of the enzyme-bound copper is expressed by a signal close to, but not identical with, that of native enzyme. Further addition of CN- induces a simultaneous decrease in intensity of both of these signals so that their 1:1 ratio is maintained. Binding of N3-, on the other hand, changes the e.p.r. spectrum to a form different from either that of the native or CN- -treated enzyme, and integrates to 100% of the copper in the enzyme (g = 2.252, g = 2.050, A = 16.5 mT). Resolved superhyperfine structure is apparent in the g region. N3- binding is also accompanied by the appearance of a broad charge-transfer band centred at 387 nm. Neither 9 nor 35 GHz e.p.r. spectra show evidence for more than one (non-interacting) species of Cu(II) in native enzyme and N3- derivatives. The binding and reactivity of CN-, on the other hand, argues against independent copper sites in the enzyme.

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Binding sites of anions in superoxide dismutase

Cited by 45 publications

References 4 publications

Phosphate is an inhibitor of copper-zinc superoxide dismutase

Phosphate is an inhibitor of copper-zinc superoxide dismutase

Cu,Zn Superoxide Dismutase from Photobacterium leiognathi Is an Hyperefficient Enzyme

Kinetic and e.p.r. studies of cyanide and azide binding to the copper sites of dopamine (3,4-dihydroxyphenethylamine) β-mono-oxygenase

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