Terentyev Vasily V, Trubitsina Liubov I, Shukshina Anna K, Trubitsin Ivan V, Rudenko Natalia N
Institute of Basic Biological Problems, Federal Research Center "Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences", 142290 Pushchino, Russia.
G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Federal Research Center "Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences", 142290 Pushchino, Russia.
Plants (Basel). 2024 Dec 27;14(1):55. doi: 10.3390/plants14010055.
The green unicellular algae contains 12-13 carbonic anhydrases (CAs). For a long time, the two closely related α-CAs of the periplasmic membrane CAH1 and CAH2 were considered to be the CAs with the highest CO hydration activity. The recombinant protein α-CA CAH3 (rCAH3) from the thylakoid lumen obtained in the present study showed more than three times higher activity compared to CAH1 and more than 11 times higher compared to previous studies with rCAH3. Long-term sustainability of the enzyme was observed at alkaline pH (>8), with maintenance of half of its activity at 4 °C for up to 50 days. Thermostability of rCAH3 indicated the retention of the activity at 20 °C for one hour at pH 9-10 with its ~50% decrease at pH 6-7. However, the residual activity of rCAH3 after incubation at an extremely high temperature (75 °C) for 15 min led to the formation of the double-hump graph with maxima at pH 6 and 9. The enzyme demonstrated high sensitivity to ethoxyzolamide and acetazolamide at nM concentrations, to Zn and Cu cations at 1 mM concentrations, and L-cysteine was able to completely inhibit CA activity of rCAH3 through reduction of sulfhydryl groups. Esterase activity of rCAH3 was well detected with values comparable to those of bovine CAII, but with a maximum at pH 8 instead of pH 9, which is usual for bovine CAII. The results indicated that CAH3 may be the most active CA of and that its role in the photosynthetic apparatus function could have been underestimated in previous works.
这种绿色单细胞藻类含有12 - 13种碳酸酐酶（CAs）。长期以来，周质膜CAH1和CAH2这两种密切相关的α - CAs被认为是具有最高CO水合活性的碳酸酐酶。本研究中从类囊体腔获得的重组蛋白α - CA CAH3（rCAH3）显示出比CAH1高3倍以上的活性，比之前对rCAH3的研究高11倍以上。在碱性pH（>8）下观察到该酶的长期稳定性，在4℃下其活性可维持一半长达50天。rCAH3的热稳定性表明，在pH 9 - 10时，其活性在20℃下可保持1小时，在pH 6 - 7时活性下降约50%。然而，rCAH3在极高温度（75℃）下孵育15分钟后的残余活性导致形成双峰图，最大值出现在pH 6和9处。该酶对纳摩尔浓度的乙氧唑胺和乙酰唑胺、1毫摩尔浓度的锌和铜阳离子表现出高敏感性，L - 半胱氨酸能够通过还原巯基完全抑制rCAH3的CA活性。rCAH3的酯酶活性检测良好，其值与牛碳酸酐酶II相当，但最大值出现在pH 8而非牛碳酸酐酶II通常的pH 9处。结果表明，CAH3可能是该藻类中活性最高的碳酸酐酶，并且其在光合装置功能中的作用在以往研究中可能被低估了。
