Changes in an Enzyme Ensemble During Catalysis Observed by High Resolution XFEL Crystallography

Authors

Nathan J. Smith, University of Nebraska-LincolnFollow
Medhanjali Dasgupta, University of Nebraska- LincolnFollow
David C. Wych, Los Alamos National Laboratory
Cole Dolamore, University of Nebraska- LincolnFollow
Raymond G. Sierra, Stanford University
Stella Lisova, Stanford University
Darya Marchany-Rivera, SLAC National Accelerator Laboratory
Aina E. Cohen, SLAC National Accelerator Laboratory
Sébastien Boutet, SLAC National Accelerator Laboratory
Mark S. Hunter, SLAC National Accelerator Laboratory
Christopher Kupitz, SLAC National Accelerator Laboratory
Frédéric Poitevin, SLAC National Accelerator Laboratory
Frank R. Moss III, SLAC National Accelerator Laboratory
Aaron S. Brewster, Lawrence Berkeley National Laboratory
Nicholas K. Sauter, Lawrence Berkeley National Laboratory
Iris D. Young, Lawrence Berkeley National Laboratory
Alexander M. Wolff, University of California, Merced
Virendra K. Tiwari, University of Nebraska-Lincoln
Nivesh Kumar, University of Nebraska-Lincoln
David B. Berkowitz, University of Nebraska - LincolnFollow
Ryan G. Hadt, California Institute of Technology
Michael C. Thompson, University of California, Merced
Alec H. Follmer, University of California-Irvine
Mark A. Wilson, Department of BiochemistryFollow

Date of this Version

8-16-2023

Citation

doi: https://doi.org/10.1101/2023.08.15.553460

Comments

Used by permission.

Abstract

Enzymes populate ensembles of structures with intrinsically different catalytic proficiencies that are difficult to experimentally characterize. We use time-resolved mix-and-inject serial crystallography (MISC) at an X-ray free electron laser (XFEL) to observe catalysis in a designed mutant (G150T) 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 catalytic intermediate selects for catalytically competent substates. A prior proposal for active site cysteine charge-coupled conformational changes in ICH is validated by determining structures of the enzyme over a range of pH values. A combination of large molecular dynamics simulations of the enzyme in crystallo and timeresolved electron density maps shows that ionization of the general acid Asp17 during catalysis causes additional conformational changes that propagate across the dimer interface, connecting the two active sites. These ionization-linked changes in the ICH conformational ensemble permit water to enter the active site in a location that is poised 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.