Structural and catalytic effects of proline substitution and surface loop deletion in the extended active site of human carbonic anhydrase II.
Structural and catalytic effects of proline substitution and surface loop deletion in the extended active site of human carbonic anhydrase II.
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DOI：
10.1111/febs.13232
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发表时间：
2015-04
期刊：
影响因子：
--
通讯作者：
McKenna R
中科院分区：
文献类型：
--
作者：
Boone CD;Rasi V;Tu C;McKenna R
从嗜温支架开始的嗜热酶的生物工程已经被证明是一个重大的挑战，因为已经提出了几种稳定元素是热稳定性的基础，包括二硫键、表面环还原、离子对网络、脯氨酸取代和芳香族簇。这项研究强调了通过在170和234位掺入脯氨酸残基来增加人碳酸酐酶II（HCA II）刚性的影响，脯氨酸残基位于能够容纳限制性主链构象而不重排周围肽骨架的表面环中。此外，通过删除表面环（残基230至240）的方式检查了HCA II的紧凑性的效果，该表面环先前已被鉴定为从细菌Sulphidohydrogenibium yellowstonense YO3AOP1分离的超嗜热CA的热稳定性的可能来源。这些HCA II变体的差示扫描量热法分析表明，这些结构修饰对酶的热稳定性的影响最小，而动力学研究显示出意想不到的催化效率和质子转移速率的影响。这些HCA II变体的X射线晶体学分析表明，高酸性环（残基230和240）的静电势和构型在其高催化活性中起着重要作用。基于这些观察和文献，HCA II的一般结构架构内的各种组件的图片正在出现，这些组件是稳定性的关键。这些元素可以为其他酶的合理热稳定性工程提供蓝图。
The bioengineering of a thermophilic enzyme starting from a mesophilic scaffold has proven to be a significant challenge as several stabilizing elements have been proposed to be the foundation of thermal stability including disulfide bridges, surface loop reduction, ionic pair networks, proline substitutions, and aromatic clusters. This study emphasizes the impact of increasing the rigidity of human carbonic anhydrase II (HCA II) via incorporation of proline residues at positions 170 and 234, which are located in surface loops that are able to accommodate restrictive main-chain conformations without rearrangement of the surrounding peptide backbone. Additionally, the effect of compactness of HCA II was examined by way of deletion of a surface loop (residues 230 through 240), which had been previously identified as a possible source of thermal stability for the hyperthermophilic CA isolated from the bacterium Sulfurihydrogenibium yellowstonense YO3AOP1. Differential scanning calorimetry analysis of these HCA II variants revealed that these structural modifications had a minimum effect on the thermal stability of the enzyme while kinetic studies showed unexpected effects on the catalytic efficiency and proton transfer rates. X-ray crystallographic analysis of these HCA II variants showed the electrostatic potential and configuration of the highly acidic loop (residues 230 and 240) plays an important role in its high catalytic activity. Based on these observations and the literature, a picture is emerging of the various components within the general structural architecture of HCA II that are key to stability. These elements could provide the blueprints for the rational thermal stability engineering of other enzymes.
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人类碳赤霉素II的活性位点中的短而强的氢键。
影响因子：
2.9
作者：
Avvaru, Balendu Sankara;Kim, Chae Un;Sippel, Katherine H.;Gruner, Sol M.;Agbandje-McKenna, Mavis;Silverman, David N.;McKenna, Robert
通讯作者：
McKenna, Robert
DOI：
10.1107/s0907444913007208
发表时间：
2013-06-01
影响因子：
2.2
作者：
Di Fiore, Anna;Capasso, Clemente;De Simone, Giuseppina
通讯作者：
De Simone, Giuseppina
影响因子：
8
作者：
Filikov, AV;Hayes, RJ;Dahiyat, BI
通讯作者：
Dahiyat, BI
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
3.9
作者：
Bonisch, H;Backmann, J;Schafer, G
通讯作者：
Schafer, G