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首页> 外文期刊>Biochemistry >Putative Functional Role for the Invariant Aspartate 263 Residue of Rhodospirillum rubrum Rubisco
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Putative Functional Role for the Invariant Aspartate 263 Residue of Rhodospirillum rubrum Rubisco

机译：假定的功能作用的鲁棒红螺菌天冬氨酸263不变残留。

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Although aspartate residue D263 of Rhodospirillum rubrum Rubisco is close to the active sitenand invariant in all reported Rubiscos, its possible functional and structural roles in Rubisco activity havennot been investigated. We have mutagenised D263 to several selected amino acids (asparagine, alanine,nserine, glutamate, and glutamine) to probe possible roles in facilitating proton movements within thenactive site and maintaining structural positioning of key active-site groups. The mutants have beenncharacterized by kinetic methods and by differential scanning calorimetry (DSC) to examine the effectsnof the substitutions on the stability of the folded state. We show that D263 is essential for maintainingneffective levels of catalysis with the mutations reducing carboxylation variously by up to 100-fold butnhaving less than 10% effect on the carboxylase/oxygenase specificity of the catalytic reaction. Removingnthe charge of the residue 263 side chain significantly strengthens binding of the activating (carbamylating)nCO2 molecule. In contrast, a charge on the 263 site has only a small influence on binding of the positivelyncharged Mg2+ ion, suggesting that the local protein structure provides different shielding of the formalncharges on the Mg2+ ion and the ε-lysine group of K191. Interestingly, introduction of an internal cavityn(D263S and D263A) and insertion of an extra -CH2- group (D263E and D263Q) have opposite effectsnon catalysis, the former relatively small and the latter much larger, suggesting that the extra side-chainngroup induces a specific structural distortion that inhibits formation of the transition state. As the DSCnresults show that the mutations only slightly increase the kinetic stability of the folded state, we concludenthat the rate-limiting (activated) step of unfolding involves substantial unfolding of the structure but notnin the region of site 263. In summary, interaction of D263 with H287 of a largely electrostatic naturenappears critical for maintaining correct positioning of catalytic groups in the active site. The conservationnof D263 can thus be accounted for by its contribution to the maintenance of a finely tuned structure innthis region abutting the active site.
机译：尽管在所有报道的Rubiscos中，Rhodospirillum rubrum Rubisco的天冬氨酸残基D263均接近活性位点和不变量，但尚未研究其在Rubisco活性中可能的功能和结构作用。我们已经将D263诱变为几个选定的氨基酸（天冬酰胺，丙氨酸，nserine，谷氨酸和谷氨酰胺），以探究促进质子在随后的活性位点内移动并维持关键活性位点组的结构定位的可能作用。已经通过动力学方法和通过差示扫描量热法（DSC）表征了突变体，以检查取代对折叠状态稳定性的影响。我们表明，D263对于维持有效的催化水平至关重要，其突变可通过最多100倍降低羧化程度的各种突变，但对催化反应的羧化酶/加氧酶的特异性影响不到10％。除去残基263侧链的电荷显着增强了活化（氨基甲酰化）nCO 2分子的结合。相反，263位上的电荷对带正电的Mg2 +离子的结合影响很小，这表明局部蛋白质结构对Mg2 +离子和K191的ε-赖氨酸基团上的福尔曼电荷提供了不同的屏蔽。有趣的是，引入内腔（D263S和D263A）和插入一个额外的-CH2-基团（D263E和D263Q）具有相反的非催化作用，前者相对较小，后者则大得多，这表明额外的侧链基团会诱导阻止过渡态形成的特定结构扭曲。由于DSCn结果显示突变仅稍微增加了折叠状态的动力学稳定性，因此我们得出结论，展开的限速（激活）步骤涉及结构的实质展开，但不在位点263的区域中。总之，D263的相互作用H287具有很大的静电性质，对于保持活性基团中催化基团的正确定位至关重要。因此，D263的保守性可以通过其对维持与活动位点邻接的区域中的微调结构的贡献来解决。

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  • 来源
    《Biochemistry》 |2009年第10期|p.2226-2236|共11页
  • 作者

    John R. Liggins and Jill E. Gready;

  • 作者单位

    Molecular Plant Physiology Group, Research School of Biological Sciences, and DiVision of Molecular Bioscience,John Curtin School of Medical Research, Australian National UniVersity, Canberra ACT 0200, Australia;

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