Changes in an enzyme ensemble during catalysis observed by high-resolution XFEL crystallography

• Published in: Science advances Vol. 10; no. 13; p. eadk7201
• Main Authors: Smith, Nathan, Dasgupta, Medhanjali, Wych, David C, Dolamore, Cole, Sierra, Raymond G, Lisova, Stella, Marchany-Rivera, Darya, Cohen, Aina E, Boutet, Sébastien, Hunter, Mark S, Kupitz, Christopher, Poitevin, Frédéric, Moss, 3rd, Frank R, Mittan-Moreau, David W, Brewster, Aaron S, Sauter, Nicholas K, Young, Iris D, Wolff, Alexander M, Tiwari, Virendra K, Kumar, Nivesh, Berkowitz, David B, Hadt, Ryan G, Thompson, Michael C, Follmer, Alec H, Wall, Michael E, Wilson, Mark A
• Format: Journal Article
• Language: English
• Published: United States AAAS 29.03.2024; American Association for the Advancement of Science
• Subjects: BASIC BIOLOGICAL SCIENCES; Biochemistry; Biomedicine and Life Sciences; Catalysis; Crystallography, X-Ray; Hydrolases; Molecular Dynamics Simulation; Protein Conformation; Proteins - chemistry; SciAdv r-articles; Structural Biology
• ISSN: 2375-2548; 2375-2548
• DOI: 10.1126/sciadv.adk7201

Enzymes populate ensembles of structures necessary for catalysis that are difficult to experimentally characterize. We use time-resolved mix-and-inject serial crystallography at an x-ray free electron laser to observe catalysis in a designed mutant isocyanide hydratase (ICH) enzyme that enhances sampling of important minor conformations. The active site exists in a mixture of conformations, and formation of the thioimidate intermediate selects for catalytically competent substates. The influence of cysteine ionization on the ICH ensemble is validated by determining structures of the enzyme at multiple pH values. Large molecular dynamics simulations in crystallo and time-resolved electron density maps show that Asp ionizes during catalysis and causes conformational changes that propagate across the dimer, permitting water to enter the active site for intermediate hydrolysis. ICH exhibits a tight coupling between ionization of active site residues and catalysis-activated protein motions, exemplifying a mechanism of electrostatic control of enzyme dynamics.