biostudies-literatureKim JKNational Research Foundation of Korea (NRF)SamsungNIGMS NIH HHS4404https://www.ebi.ac.uk/biostudies/studies/S-EPMC12069641biostudies-literatureUnknown16(1)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 (CO₂) 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 activeNature communicationsFast product release requires active-site water dynamics in carbonic anhydrase.PMC12069641RS-2024-00353098RS-2022-NR0704632021R1C1C1010943P30 GM133894P30 GM124166SSTF-BA1702-042021R1A5A80294902020R1C1C1003937Choi JMJeong HCohen AEJo BHKim JKLomelino CGhim CMWierman JLShin TJKumar RKim SAndring JTLee CSon DWKim CUMin DMcKenna RLim SWfalseFast product release requires active-site water dynamics in carbonic anhydrase.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 (CO₂) 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 active2025-01-01T00:00:00Z2025 May2026-06-03T02:44:56.406Z2026-04-23T03:11:01.839ZS-EPMC120696414035544010.1038/s41467-025-59645-x