To investigate the structural/functional role of the dimeric structure in Cu,Zn superoxide dismutases, we have studied the stability to a variety of agents of the Escherichia coli enzyme, the only monomeric variant of this class so far isolated, Differential scanning calorimetry of the native enzyme showed the presence of two well defined peaks identified as the metal free and holoprotein, Unlike dimeric Cu,Zn superoxide dismutases, the unfolding of the monomeric enzyme was found to be highly reversible, a behavior that may be explained by the absence of free cysteines and the highly polar nature of its molecular surface. The melting temperature of the E. coli enzyme was found to be pH-dependent with the holoenzyme transition centered at 66 degrees C at pH 7.8 and at 79.3 degrees C at pH 6.0, The active-site metals, which were easily displaced from the active site by EDTA, were found to enhance the thermal stability of the monomeric apoprotein but to a lower extent than in the dimeric enzymes from eukaryotic sources, Apo-superoxide dismutase from E. coli was shown to be nearly as stable as the bovine apoenzyme, whose hole form is much more stable and less sensitive to pH variations. The remarkable pH susceptibility of the E. coli enzyme structure was paralleled by the slow decrease in activity of the enzyme incubated at alkaline pH and by modification of the EPR spectrum at lower pH values than in the case of dimeric enzymes, Unlike eukaryotic Cu,Zn superoxide dismutases, the active-site structure of the E. coli enzyme was shown to be reversibly perturbed by urea, These observations suggest that the conformational stability of Cu,Zn superoxide dismutases is largely due to the intrinsic stability of the beta-barrel fold rather than to the dimeric structure and that pH sensitivity and weak metal binding of the E. coli enzyme are due to higher flexibility and accessibility to the solvent of its active-site region.

Battistoni, A., Folcarelli, S., Cervoni, L., Polizio, F., Desideri, A., Giartosio, A., et al. (1998). Role of the dimeric structure in Cu,Zn superoxide dismutase - pH-dependent, reversible denaturation of the monomeric enzyme from Escherichia coli. THE JOURNAL OF BIOLOGICAL CHEMISTRY, 273(10), 5655-5661 [10.1074/jbc.273.10.5655].

Role of the dimeric structure in Cu,Zn superoxide dismutase - pH-dependent, reversible denaturation of the monomeric enzyme from Escherichia coli

BATTISTONI, ANDREA;POLIZIO, FRANCESCA;DESIDERI, ALESSANDRO;ROTILIO, GIUSEPPE
1998-01-01

Abstract

To investigate the structural/functional role of the dimeric structure in Cu,Zn superoxide dismutases, we have studied the stability to a variety of agents of the Escherichia coli enzyme, the only monomeric variant of this class so far isolated, Differential scanning calorimetry of the native enzyme showed the presence of two well defined peaks identified as the metal free and holoprotein, Unlike dimeric Cu,Zn superoxide dismutases, the unfolding of the monomeric enzyme was found to be highly reversible, a behavior that may be explained by the absence of free cysteines and the highly polar nature of its molecular surface. The melting temperature of the E. coli enzyme was found to be pH-dependent with the holoenzyme transition centered at 66 degrees C at pH 7.8 and at 79.3 degrees C at pH 6.0, The active-site metals, which were easily displaced from the active site by EDTA, were found to enhance the thermal stability of the monomeric apoprotein but to a lower extent than in the dimeric enzymes from eukaryotic sources, Apo-superoxide dismutase from E. coli was shown to be nearly as stable as the bovine apoenzyme, whose hole form is much more stable and less sensitive to pH variations. The remarkable pH susceptibility of the E. coli enzyme structure was paralleled by the slow decrease in activity of the enzyme incubated at alkaline pH and by modification of the EPR spectrum at lower pH values than in the case of dimeric enzymes, Unlike eukaryotic Cu,Zn superoxide dismutases, the active-site structure of the E. coli enzyme was shown to be reversibly perturbed by urea, These observations suggest that the conformational stability of Cu,Zn superoxide dismutases is largely due to the intrinsic stability of the beta-barrel fold rather than to the dimeric structure and that pH sensitivity and weak metal binding of the E. coli enzyme are due to higher flexibility and accessibility to the solvent of its active-site region.
1998
Pubblicato
Rilevanza internazionale
Articolo
Sì, ma tipo non specificato
Settore BIO/10 - BIOCHIMICA
English
Con Impact Factor ISI
apoenzyme; copper zinc superoxide dismutase; cysteine; dimer; edetic acid; metal ion; monomer; solvent; article; differential scanning calorimetry; enzyme active site; enzyme activity; enzyme binding; enzyme denaturation; enzyme stability; enzyme structure; escherichia coli; metal binding; nonhuman; ph; priority journal; Apoenzymes; Binding Sites; Calorimetry, Differential Scanning; Dimerization; Electron Spin Resonance Spectroscopy; Enzyme Stability; Escherichia coli; Hydrogen-Ion Concentration; Protein Conformation; Protein Folding; Superoxide Dismutase; Temperature; Thermodynamics; Urea; Bacteria (microorganisms); Bovinae; Escherichia coli; Eukaryota
Battistoni, A., Folcarelli, S., Cervoni, L., Polizio, F., Desideri, A., Giartosio, A., et al. (1998). Role of the dimeric structure in Cu,Zn superoxide dismutase - pH-dependent, reversible denaturation of the monomeric enzyme from Escherichia coli. THE JOURNAL OF BIOLOGICAL CHEMISTRY, 273(10), 5655-5661 [10.1074/jbc.273.10.5655].
Battistoni, A; Folcarelli, S; Cervoni, L; Polizio, F; Desideri, A; Giartosio, A; Rotilio, G
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2108/55124
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