ROLE OF THE DIMERIC STRUCTURE IN CU,ZN SUPEROXIDE-DISMUTASE - PH-DEPENDENT, REVERSIBLE DENATURATION OF THE MONOMERIC ENZYME FROM ESCHERICHIA-COLI

To investigate the structural/functional role of the dimeric structure in Cu,Zn superoxide dismutases, we have studied the stability to a va riety of agents of the Escherichia coli enzyme, the only monomeric var iant of this class so far isolated, Differential scanning calorimetry of the native enzyme showed the presence of two well defined peaks ide ntified as the metal free and holoprotein, Unlike dimeric Cu,Zn supero xide 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-depen dent 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 e asily displaced from the active site by EDTA, were found to enhance th e thermal stability of the monomeric apoprotein but to a lower extent than in the dimeric enzymes from eukaryotic sources, Apo-superoxide di smutase from E. coli was shown to be nearly as stable as the bovine ap oenzyme, whose hole form is much more stable and less sensitive to pH variations. The remarkable pH susceptibility of the E. coli enzyme str ucture was paralleled by the slow decrease in activity of the enzyme i ncubated at alkaline pH and by modification of the EPR spectrum at low er pH values than in the case of dimeric enzymes, Unlike eukaryotic Cu ,Zn superoxide dismutases, the active-site structure of the E. coli en zyme was shown to be reversibly perturbed by urea, These observations suggest that the conformational stability of Cu,Zn superoxide dismutas es is largely due to the intrinsic stability of the beta-barrel fold r ather than to the dimeric structure and that pH sensitivity and weak m etal binding of the E. coli enzyme are due to higher flexibility and a ccessibility to the solvent of its active-site region.