Bonaccorsi di Patti M C, Carrì M T, Gabbianelli R, Da Gai R, Volpe C, Giartosio A, Rotilio G, Battistoni A
Department of Biochemical Sciences A. Rossi Fanelli and CNR Center of Molecular Biology, University of Rome La Sapienza, Italy.
Arch Biochem Biophys. 2000 May 15;377(2):284-9. doi: 10.1006/abbi.2000.1786.
The two Cu,Zn superoxide dismutases from the amphibian Xenopus laevis (denoted XSODA and XSODB) display different heat sensitivities, XSODA being more thermolabile than XSODB. In this study, we have investigated the contribution of a free cysteine residue located close to the subunit interface of XSODA to its lower thermal stability. We have found that mutation of residue Cys 150 to Ala in XSODA makes the thermal stability of this enzyme comparable to that of the wild-type XSODB isoenzyme, while the introduction of a cysteine residue in the same position of XSODB renders this enzyme variant much more heat-sensitive. Differential scanning calorimetry experiments showed that XSODA has a melting temperature about 8.5 degrees C lower than that of XSODB. On the contrary, the melting temperature of XSODACys150Ala is very close to that of XSODB, while the melting temperature of XSODBSer150Cys is even lower than that of wild-type XSODA. These data indicate that the free cysteine residue present in XSODA affects not only the reversibility of unfolding of the enzyme but also its conformational stability. We suggest that the large effect of the Cys 150 residue on XSODA stability might be due to incorrect disulfide bond formation or disulfide bond interchange during heat-induced unfolding rather than to alteration of the interaction between the enzyme subunits.
来自两栖动物非洲爪蟾的两种铜锌超氧化物歧化酶（分别记为XSODA和XSODB）表现出不同的热敏感性，XSODA比XSODB更不耐热。在本研究中，我们探究了XSODA亚基界面附近一个游离半胱氨酸残基对其较低热稳定性的影响。我们发现，将XSODA中的半胱氨酸150残基突变为丙氨酸后，该酶的热稳定性与野生型XSODB同工酶相当，而在XSODB的相同位置引入一个半胱氨酸残基则使该酶变体对热更加敏感。差示扫描量热法实验表明，XSODA的解链温度比XSODB低约8.5摄氏度。相反，XSODACys150Ala的解链温度与XSODB非常接近，而XSODBSer150Cys的解链温度甚至比野生型XSODA还要低。这些数据表明，XSODA中存在的游离半胱氨酸残基不仅影响酶解折叠的可逆性，还影响其构象稳定性。我们认为，半胱氨酸150残基对XSODA稳定性的巨大影响可能是由于热诱导解折叠过程中形成了错误的二硫键或二硫键互换，而不是由于酶亚基之间相互作用的改变。
