FASTER SUPEROXIDE-DISMUTASE MUTANTS DESIGNED BY ENHANCING ELECTROSTATIC GUIDANCE
FASTER SUPEROXIDE-DISMUTASE MUTANTS DESIGNED BY ENHANCING ELECTROSTATIC GUIDANCE
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DOI：
10.1038/358347a0
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发表时间：
1992-07-23
期刊：
影响因子：
64.8
通讯作者：
HALLEWELL, RA
中科院分区：
文献类型：
--
作者：
GETZOFF, ED;CABELLI, DE;HALLEWELL, RA
酶铜，锌超氧化物歧化酶（SOD）通过歧化超氧化物自由基O2，保护免受氧化损伤。在受扩散速率限制3，6并受静电导向增强的反应中，在活性位点铜离子4，5处与分子氧和过氧化氢1 -3反应7 -10。SOD已经发展成为已知的最快的酶之一（V（max）大约2 x 10（9）M-1 s-1）6，11。人SOD 12的新晶体结构表明，与静电导向有关的氨基酸位点链（Glu 132、Glu 133和Lys 136）形成了氢键网络。在这里，我们表明，增加局部正电荷，同时保持这种定向网络（Glu -> Gln）的位点特异性突变体具有更快的反应速率和增加离子强度的依赖性，匹配布朗动力学模拟纳入静电条款。仅仅增加正电荷是不够的：一个电荷反转（Glu --> Lys）突变体比等效的电荷中和（Glu --> Gln）突变体慢，表明新引入的正电荷破坏了定向网络。因此，静电促进的扩散速率可以通过设计来增加，只要保持活性位点静电网络的详细结构完整性。
THE enzyme Cu, Zn superoxide dismutase (SOD) protects against oxidative damage by dismuting the superoxide radical O2.- to molecular oxygen and hydrogen peroxide1-3 at the active-site Cu ion4,5 in a reaction that is rate-limited by diffusion3,6 and enhanced by electrostatic guidance7-10. SOD has evolved to be one of the fastest enzymes known (V(max) approximately 2 x 10(9) M-1 s-1)6,11. The new crystal structures of human SOD12 show that amino-acid site chains that are implicated in electrostatic guidance8 (Glu 132, Glu 133 and Lys 136) form a hydrogen-bonding network. Here we show that site-specific mutants that increase local positive charge while maintaining this orienting network (Glu --> Gln) have faster reaction rates and increased ionic-strength dependence, matching brownian dynamics simulations incorporating electrostatic terms. Increased positive charge alone is insufficient: one charge reversal (Glu --> Lys) mutant is slower than the equivalent charge neutralization (Glu --> Gln) mutant, showing that the newly introduced positive charge disrupts the orienting network. Thus, electrostatically facilitated diffusion rates can be increased by design, provided the detailed structural integrity of the active-site electrostatic network is maintained.