Battistoni A, Folcarelli S, Cervoni L, Polizio F, Desideri A, Giartosio A, Rotilio G
Department of Biology, University of Rome "Tor Vergata," Via della Ricerca Scientifica, 00133 Roma, Italy.
J Biol Chem. 1998 Mar 6;273(10):5655-61. doi: 10.1074/jbc.273.10.5655.
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 holo 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.
为了研究二聚体结构在铜锌超氧化物歧化酶中的结构/功能作用，我们研究了大肠杆菌酶（迄今为止分离出的该类唯一单体变体）对多种试剂的稳定性。天然酶的差示扫描量热法显示存在两个明确的峰，分别确定为无金属形式和全蛋白形式。与二聚体铜锌超氧化物歧化酶不同，单体酶的解折叠被发现是高度可逆的，这种行为可能是由于没有游离半胱氨酸及其分子表面的高极性性质。发现大肠杆菌酶的解链温度依赖于pH值，全酶转变在pH 7.8时以66℃为中心，在pH 6.0时以79.3℃为中心。活性位点金属很容易被EDTA从活性位点置换出来，发现它们能增强单体脱辅基蛋白的热稳定性，但程度低于真核来源的二聚体酶。大肠杆菌的脱辅基超氧化物歧化酶被证明几乎与牛脱辅基酶一样稳定，牛脱辅基酶的全酶形式更稳定且对pH变化不太敏感。大肠杆菌酶结构对pH的显著敏感性与在碱性pH下孵育的酶活性缓慢下降以及在比二聚体酶更低的pH值下EPR光谱的改变相平行。与真核铜锌超氧化物歧化酶不同，大肠杆菌酶的活性位点结构被证明会被尿素可逆地扰动。这些观察结果表明，铜锌超氧化物歧化酶的构象稳定性很大程度上归因于β-桶折叠的固有稳定性而非二聚体结构，并且大肠杆菌酶的pH敏感性和弱金属结合是由于其活性位点区域对溶剂具有更高的灵活性和可及性。
