A Single Outer-Sphere Mutation Stabilizes apo-Mn Superoxide Dismutase by 35 °C and Disfavors Mn Binding.
A Single Outer-Sphere Mutation Stabilizes apo-Mn Superoxide Dismutase by 35 °C and Disfavors Mn Binding.
复制标题
单个外球突变可将 apo-Mn 超氧化物歧化酶稳定在 35 °C 且不利于 Mn 结合。
DOI：
10.1021/acs.biochem.7b00175
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发表时间：
2017
期刊：
影响因子：
2.9
通讯作者：
Wang,Ting
中科院分区：
文献类型：
--
作者：
Miller,Anne-Frances;Wang,Ting
锰特异性超氧化物歧化酶（MnSOD）的催化活性位点围绕氧化还原活性Mn离子组织。MnSOD和同源FeSOD之间最保守的差异是第二配位球中Gln的起源。在MnSOD中，它来自C-末端结构域，而在FeSOD中，它来自N-末端结构域，但其侧链在这两种类型的SOD的活性位点中占据几乎可重叠的位置。在大肠杆菌FeSOD中将该Gln 69突变为Glu使Fe 3 +/2+还原中点电位增加> 0.6V，而不破坏结构或Fe结合[Yikilmaz，E.，Rodgers，D. W.，和米勒，A.- F.（2006）Biochemistry 45（4），1151 - 1161]。我们现在描述类似的Q146 E突变的MnSOD，解释其低锰含量的apo-Mn蛋白的稳定性增加。在0.8M盐酸胍溶液中，Q146 E-apoMnSOD的表观熔点比野生型apoMnSOD高35 ° C，而野生型apoMnSOD的表观熔点仅比野生型apoMnSOD高20 °C。而Q146 E-holoMnSOD的Tmatch比Q146 E-apoMnSOD低40 ° C。因此，我们的数据反驳了WT残基优化蛋白质结构稳定性的观点，而是与基于酶功能的保守性一致，因此与金属离子结合的能力一致。我们建议，WT-MnSOD蛋白在位置146处保存一个不稳定的氨基酸，作为有利于金属离子结合的策略的一部分。
The catalytic active site of Mn-specific superoxide dismutase (MnSOD) is organized around a redox-active Mn ion. The most highly conserved difference between MnSODs and the homologous FeSODs is the origin of a Gln in the second coordination sphere. In MnSODs it derives from the C-terminal domain whereas in FeSODs it derives from the N-terminal domain, yet its side chain occupies almost superimposable positions in the active sites of these two types of SODs. Mutation of this Gln69 to Glu inEscherichia coliFeSOD increased the Fe3+/2+reduction midpoint potential by >0.6 V without disrupting the structure or Fe binding [Yikilmaz, E., Rodgers, D. W., and Miller, A.-F. (2006) Biochemistry 45 (4), 1151−1161]. We now describe the analogous Q146E mutant of MnSOD, explaining its low Mn content in terms increased stability of the apo-Mn protein. In 0.8 M guanidinium HCl, Q146E-apoMnSOD displays an apparent melting midpoint temperature (Tm) 35 °Chigherthat of wild-type (WT) apoMnSOD, whereas theTmof WT-holoMnSOD is only 20 °C higher than that of WT-apoMnSOD. In contrast, theTmattributed to Q146E-holoMnSOD is 40 °Clowerthan that of Q146E-apoMnSOD. Thus, our data refute the notion that the WT residues optimize the structural stability of the protein and instead are consistent with conservation on the basis of enzyme function and therefore ability to bind metal ion. We propose that the WT-MnSOD protein conserves a destabilizing amino acid at position 146 as part of a strategy to favor metal ion binding.