Lim Seon Woo, Jeong Hannah, Kim Gwang Ho, Min Duyoung, Kim Jin Kyun, Kim Chae Un
Department of Physics, Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Republic of Korea.
Department of Chemistry, Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Republic of Korea.
Mol Cells. 2025 Jul;48(7):100226. doi: 10.1016/j.mocell.2025.100226. Epub 2025 May 27.
Carbonic anhydrase II (CAII) is one of the most efficient enzymes known, catalyzing the reversible hydration of CO to regulate pH and facilitate CO transport in biological systems. Its exceptional catalytic rate depends on a highly ordered active site composed of a Zn ion and a hydrogen-bonded water network that supports substrate binding, proton transfer, and product release. Among the residues maintaining this network, Thr200 plays a crucial role by stabilizing key water molecules. To investigate the structural and functional consequences of perturbing this network, we examined the T200H mutant of CAII using high-pressure cryocooling and X-ray crystallography under CO pressures of 0, 5, and 20 atm. The crystallographic snapshots captured the resting (T200H-0atm), substrate-bound (T200H-20atm), and product-bound (T200H-5atm) states of the T200H mutant. In the resting state, His200 disrupts the active site by displacing essential water molecules (W1 and W2), thereby impairing the proton transfer pathway. However, the substrate- and product-bound states reveal that His200 exhibits conformational flexibility, allowing partial restoration of the water network required for catalysis. These findings suggest that His200 functions as a dynamic gatekeeper, modulating access of water, substrate, and product to the active site. This structural plasticity explains how the T200H mutant retains partial catalytic activity despite a mutation that would otherwise severely hinder function. Our results provide new insights into active-site dynamics in CAII and offer a foundation for designing isoform-specific inhibitors or engineered carbonic anhydrase variants with tunable catalytic properties.
碳酸酐酶II（CAII）是已知效率最高的酶之一，催化CO的可逆水合作用，以调节pH值并促进生物系统中的CO运输。其卓越的催化速率取决于由锌离子和支持底物结合、质子转移及产物释放的氢键水网络组成的高度有序的活性位点。在维持该网络的残基中，苏氨酸200（Thr200）通过稳定关键水分子发挥着至关重要的作用。为了研究扰乱该网络的结构和功能后果，我们在0、5和20个大气压的CO压力下，使用高压冷冻冷却和X射线晶体学对CAII的T200H突变体进行了研究。晶体学快照捕捉到了T200H突变体的静止（T200H - 0atm）、底物结合（T200H - 20atm）和产物结合（T200H - 5atm）状态。在静止状态下，组氨酸200（His200）通过取代必需的水分子（W1和W2）破坏活性位点，从而损害质子转移途径。然而，底物结合和产物结合状态表明，His200表现出构象灵活性，允许部分恢复催化所需的水网络。这些发现表明，His200作为一个动态守门人，调节水、底物和产物进入活性位点。这种结构可塑性解释了尽管发生了否则会严重阻碍功能的突变，T200H突变体如何仍保留部分催化活性。我们的结果为CAII活性位点动力学提供了新的见解，并为设计亚型特异性抑制剂或具有可调催化特性的工程化碳酸酐酶变体提供了基础。
