Aggarwal Mayank, Chua Teck Khiang, Pinard Melissa A, Szebenyi Doletha M, McKenna Robert
Division of Biology and Soft Matter, Oak Ridge National Laboratory , Oak Ridge, Tennessee 37831, United States.
Cornell High Energy Synchrotron Source (CHESS), Cornell University , Ithaca, New York 14853, United States.
Biochemistry. 2015 Nov 3;54(43):6631-8. doi: 10.1021/acs.biochem.5b00987. Epub 2015 Oct 16.
Carbonic anhydrases (CAs) are enzymes that catalyze the hydration/dehydration of CO2/HCO3(-) with rates approaching diffusion-controlled limits (kcat/KM ∼ 10(8) M(-1) s(-1)). This family of enzymes has evolved disparate protein folds that all perform the same reaction at near catalytic perfection. Presented here is a structural study of a β-CA (psCA3) expressed in Pseudomonas aeruginosa, in complex with CO2, using pressurized cryo-cooled crystallography. The structure has been refined to 1.6 Å resolution with R(cryst) and R(free) values of 17.3 and 19.9%, respectively, and is compared with the α-CA, human CA isoform II (hCA II), the only other CA to have CO2 captured in its active site. Despite the lack of structural similarity between psCA3 and hCA II, the CO2 binding orientation relative to the zinc-bound solvent is identical. In addition, a second CO2 binding site was located at the dimer interface of psCA3. Interestingly, all β-CAs function as dimers or higher-order oligomeric states, and the CO2 bound at the interface may contribute to the allosteric nature of this family of enzymes or may be a convenient alternative binding site as this pocket has been previously shown to be a promiscuous site for a variety of ligands, including bicarbonate, sulfate, and phosphate ions.
碳酸酐酶(CAs)是一类能催化CO₂/HCO₃⁻水合/脱水反应的酶,其反应速率接近扩散控制极限(kcat/KM ∼ 10⁸ M⁻¹ s⁻¹)。该酶家族进化出了不同的蛋白质折叠结构,但都能以近乎完美的催化效率进行相同的反应。本文利用加压低温冷却晶体学技术,对铜绿假单胞菌中表达的一种β - 碳酸酐酶(psCA3)与CO₂形成的复合物进行了结构研究。该结构已精修至1.6 Å分辨率,R(cryst)和R(free)值分别为17.3%和19.9%,并与α - 碳酸酐酶、人碳酸酐酶同工酶II(hCA II)进行了比较,hCA II是唯一一种活性位点捕获了CO₂的其他碳酸酐酶。尽管psCA3和hCA II在结构上缺乏相似性,但相对于锌结合溶剂的CO₂结合方向是相同的。此外,在psCA3的二聚体界面处发现了第二个CO₂结合位点。有趣的是,所有β - 碳酸酐酶均以二聚体或更高阶寡聚体状态发挥作用,结合在界面处的CO₂可能有助于该酶家族的变构性质,或者可能是一个方便的替代结合位点,因为此前已表明该口袋对包括碳酸氢根、硫酸根和磷酸根离子在内的多种配体具有广泛的结合能力。