Fisher C L, Cabelli D E, Hallewell R A, Beroza P, Lo T P, Getzoff E D, Tainer J A
Department of Molecular Biology, Scripps Research Institute, La Jolla, California 92037, USA.
Proteins. 1997 Sep;29(1):103-12. doi: 10.1002/(sici)1097-0134(199709)29:1<103::aid-prot8>3.0.co;2-g.
Key charged residues in Cu,Zn superoxide dismutase (Cu,Zn SOD) promote electrostatic steering of the superoxide substrate to the active site Cu ion, resulting in dismutation of superoxide to oxygen and hydrogen peroxide, Lys-136, along with the adjacent residues Glu-132 and Glu-133, forms a proposed electrostatic triad contributing to substrate recognition. Human Cu,Zn SODs with single-site replacements of Lys-136 by Arg,Ala, Gln, or Glu or with a triple-site substitution (Glu-132 and Glu-133 to Gln and Lys-136 to Ala) were made to test hypotheses regarding contributions of these residues to Cu,Zn SOD activity. The structural effects of these mutations were modeled computationally and validated by the X-ray crystallographic structure determination of Cu,Zn SOD having the Lys-136-to-Glu replacement. Brownian dynamics simulations and multiple-site titration calculations predicted mutant reaction rates as well as ionic strength and pH effects measured by pulse-radiolytic experiments. Lys-136-to-Glu charge reversal decreased dismutation activity 50% from 2.2 x 10(9) to 1.2 x 10(9) M-1 s-1 due to repulsion of negatively charged superoxide, whereas charge-neutralizing substitutions (Lys-136 to Gln or Ala) had a less dramatic influence. In contrast, the triple-mutant Cu,Zn SOD (all three charges in the electrostatic triad neutralized) surprisingly doubled the reaction rate compared with wild-type enzyme but introduced phosphate inhibition. Computational and experimental reaction rates decreased with increasing ionic strength in all of the Lys-136 mutants, with charge reversal having a more pronounced effect than charge neutralization, implying that local electrostatic effects still govern the dismutation rates. Multiple-site titration analysis showed that deprotonation events throughout the enzyme are likely responsible for the gradual decrease in SOD activity above pH 9.5 and predicted a pKa value of 11.7 for Lys-136. Overall, Lys-136 and Glu-132 make comparable contributions to substrate recognition but are less critical to enzyme function than Arg-143, which is both mechanistically and electrostatically essential. Thus, the sequence-conserved residues of this electrostatic triad are evidently important solely for their electrostatic properties, which maintain the high catalytic rate and turnover of Cu,Zn SOD while simultaneously providing specificity by selecting against binding by other anions.
铜锌超氧化物歧化酶（Cu,Zn SOD）中的关键带电残基促进超氧化物底物向活性位点铜离子的静电导向，导致超氧化物歧化为氧气和过氧化氢。赖氨酸-136与相邻残基谷氨酸-132和谷氨酸-133形成一个推测的静电三联体，有助于底物识别。构建了用精氨酸、丙氨酸、谷氨酰胺或谷氨酸单点取代赖氨酸-136的人Cu,Zn SOD，或进行三点取代（谷氨酸-132和谷氨酸-133替换为谷氨酰胺，赖氨酸-136替换为丙氨酸），以检验这些残基对Cu,Zn SOD活性贡献的假设。通过对赖氨酸-136替换为谷氨酸的Cu,Zn SOD进行X射线晶体学结构测定，对这些突变的结构效应进行了计算建模和验证。布朗动力学模拟和多位点滴定计算预测了突变体的反应速率以及脉冲辐射实验测量的离子强度和pH效应。赖氨酸-136替换为谷氨酸的电荷反转使歧化活性从2.2×10⁹降至1.2×10⁹ M⁻¹ s⁻¹，降低了50%，这是由于带负电的超氧化物之间的排斥作用，而电荷中和取代（赖氨酸-136替换为谷氨酰胺或丙氨酸）的影响较小。相比之下，三联体突变的Cu,Zn SOD（静电三联体中的所有三个电荷均被中和）与野生型酶相比，反应速率惊人地提高了一倍，但引入了磷酸盐抑制作用。在所有赖氨酸-136突变体中，计算和实验得到的反应速率均随离子强度的增加而降低，电荷反转的影响比电荷中和更显著，这意味着局部静电效应仍然决定着歧化速率。多位点滴定分析表明，酶整体上的去质子化事件可能是导致pH 9.5以上SOD活性逐渐降低的原因，并预测赖氨酸-136的pKa值为11.7。总体而言，赖氨酸-136和谷氨酸-132对底物识别的贡献相当，但对酶功能的重要性低于精氨酸-143，精氨酸-143在机制和静电方面都是必不可少的。因此，这个静电三联体中序列保守的残基显然仅因其静电性质而重要，这些性质维持了Cu,Zn SOD的高催化速率和周转率，同时通过阻止其他阴离子的结合提供特异性。
