Kim Jin Kyun, Lim Seon Woo, Jeong Hannah, Lee Cheol, Kim Seoyoon, Son Dong Woo, Kumar Rajeev, Andring Jacob T, Lomelino Carrie, Wierman Jennifer L, Cohen Aina E, Shin Tae Joo, Ghim Cheol-Min, McKenna Robert, Jo Byung Hoon, Min Duyoung, Choi Jeong-Mo, Kim Chae Un
Department of Physics, Ulsan National Institute of Science and Technology (UNIST), Ulsan, 44919, Republic of Korea.
Frontier of Extreme Physics, Korea Research Institute of Standards and Science, Daejeon, 34113, Republic of Korea.
Nat Commun. 2025 May 12;16(1):4404. doi: 10.1038/s41467-025-59645-x.
Water plays an essential role in enzyme structure, stability, and the substantial rate enhancement of enzyme catalysis. However, direct observations linking enzyme catalysis and active-site water dynamics pose a significant challenge due to experimental difficulties. By integrating an ultraviolet (UV) photolysis technique with temperature-controlled X-ray crystallography, we track the catalytic pathway of carbonic anhydrase II (CAII) at 1.2 Å resolution. This approach enables us to construct molecular movies of CAII catalysis, encompassing substrate (CO) binding, conversion from substrate to product (bicarbonate), and product release. In the catalytic pathway, we identify an unexpected configuration in product binding and correlate it with sub-nanosecond rearrangement of active-site water. Based on these experimental observations, we propose a comprehensive mechanism of CAII and describe the detailed structure and dynamics of active-site water in CAII. Our findings suggest that CAII has evolved to utilize the structure and fast dynamics of the active-site waters for its diffusion-limited catalytic efficiency.
水在酶的结构、稳定性以及酶催化作用显著的速率增强方面发挥着至关重要的作用。然而，由于实验上的困难，将酶催化作用与活性位点水动力学联系起来的直接观察面临重大挑战。通过将紫外线（UV）光解技术与温度控制的X射线晶体学相结合，我们以1.2埃的分辨率追踪碳酸酐酶II（CAII）的催化途径。这种方法使我们能够构建CAII催化作用的分子电影，涵盖底物（CO）结合、从底物到产物（碳酸氢盐）的转化以及产物释放。在催化途径中，我们在产物结合中识别出一种意想不到的构型，并将其与活性位点水的亚纳秒重排相关联。基于这些实验观察结果，我们提出了CAII的全面机制，并描述了CAII中活性位点水的详细结构和动力学。我们的研究结果表明，CAII已经进化到利用活性位点水的结构和快速动力学来实现其扩散限制的催化效率。
