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首页> 外文期刊>Journal of enzyme inhibition and medicinal chemistry. >A failed tentative to design a super carbonic anhydrase having the biochemical properties of the most thermostable CA (SspCA) and the fastest (SazCA) enzymes
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A failed tentative to design a super carbonic anhydrase having the biochemical properties of the most thermostable CA (SspCA) and the fastest (SazCA) enzymes

机译：尝试设计具有最热稳定的CA（SspCA）和最快的（SazCA）酶的生化特性的超碳酸酐酶

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摘要

Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze a simple reaction in all life domains: the carbon dioxide hydration to bicarbonate and protons: CO2?+?H2O?→ +?H+. Six different, genetically distinct CA families are known to date, the α-, β-, γ-, δ-, ζ- and η-CAs. Bacteria encode for CAs belong to the α-, β- and γ-classes. Recently, our groups investigated the presence of CAs in two bacteria belonging to the genus Sulfurihydrogenibium living in hot springs all over the world, at temperatures of up to 110 °C. The α-CAs from Sulfurihydrogenibium yellowstonense and Sulfurihydrogenibium azorense, denominated SspCA and SazCA, respectively, are highly thermostable, maintaining a good catalytic activity even after being heated for a prolonged period. Moreover, SazCA was to be the fastest CA known to date with a kcat value of 4.40 × 106 s?1 and a kcat/KM value of 3.5 × 108 M?1 s?1. SspCA also showed a good catalytic activity for the same reaction, with a kcat value of 9.35 × 105 s?1 and a kcat/KM value of 1.1 × 108 M?1 s?1, proving that the “extremo-α-CAs” are between the most effective CAs known to date. Here, we describe a failed tentative to obtain a super-CA, SupCA, by combining the amino acid sequence of SazCA and SspCA. To achieve this goal we introduced six His residues in N-terminal sequence of SspCA. However the obtained SupCA showed lower catalytic activity and thermostability compared to both extremophilic enzymes from which it has been designed. We rationalized the biochemical reasons of this failure, which may be useful to design enzymes with a better catalytic activity.
机译：碳酸酐酶（CAs，EC 4.2.1.1）在所有生命域中催化一个简单的反应：二氧化碳水合成碳酸氢根和质子：CO 2 ？+？H 2 O ？→+？H + 。迄今为止已知六个不同的，遗传上不同的CA家族，即α-，β-，γ-，δ-，ζ-和η-CA。编码CA的细菌属于α-，β-和γ类。最近，我们的研究小组调查了生活在世界各地温泉中，温度高达110°C的两种属于硫氢化氢属的细菌中CA的存在。分别来自Sulfurihydrogenibium yellowstonense和Sulfurihydrogenibium azorense的α-CAs高度热稳定，即使长时间加热也保持良好的催化活性。此外，SazCA是迄今为止已知的最快的CA，其ak cat 值为4.40×10 6 s ？1 和ak cat / K M 值为3.5×10 8 M ？1 s ？1 。 SspCA对同样的反应也显示出良好的催化活性，ak cat 值为9.35×10 5 s ？1 和ak cat / K M 值为1.1×10 8 M ？1 s ？1 “极端-α-CA”位于迄今为止已知的最有效的CA之间。在这里，我们描述了通过结合SazCA和SspCA的氨基酸序列来获得超级CA SupCA的失败尝试。为了实现该目标，我们在SspCA的N末端序列中引入了六个His残基。然而，所获得的SupCA与设计它的两种极端酶相比均显示出较低的催化活性和热稳定性。我们合理化了此失败的生化原因，这可能对设计具有更好催化活性的酶可能有用。

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