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Proc Natl Acad Sci U S A. 2019 Dec 17;116(51):25634-25640. doi: 10.1073/pnas.1901864116. Epub 2019 Dec 4.

Mix-and-inject XFEL crystallography reveals gated conformational dynamics during enzyme catalysis.

Author information

1
Department of Biochemistry and Redox Biology Center, University of Nebraska, Lincoln, NE 68588.
2
Chair of Applied Dynamics, Friedrich-Alexander University Erlangen-Nürnberg, 91058 Erlangen, Germany.
3
Bioengineering Department, Stanford University, Stanford, CA 94305.
4
Bioscience Division, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, CA 94025.
5
Chemistry Department, University of Puerto Rico, Mayagüez PR 00681.
6
Linac Coherent Light Source, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, CA 94025.
7
Stanford PULSE Institute, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, CA 94025.
8
Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, CA 94025.
9
Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720.
10
Department of Chemistry, University of Nebraska, Lincoln, NE 68588.
11
Department of Bioengineering and Therapeutic Sciences, California Institute for Quantitative Biology, University of California, San Francisco, CA 94158.
12
Computer, Computational, and Statistical Sciences Division, Los Alamos National Laboratory, Los Alamos, NM 87505.
13
Bioscience Division, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, CA 94025; vdbedem@stanford.edu mwilson13@unl.edu.
14
Department of Biochemistry and Redox Biology Center, University of Nebraska, Lincoln, NE 68588; vdbedem@stanford.edu mwilson13@unl.edu.

Abstract

How changes in enzyme structure and dynamics facilitate passage along the reaction coordinate is a fundamental unanswered question. Here, we use time-resolved mix-and-inject serial crystallography (MISC) at an X-ray free electron laser (XFEL), ambient-temperature X-ray crystallography, computer simulations, and enzyme kinetics to characterize how covalent catalysis modulates isocyanide hydratase (ICH) conformational dynamics throughout its catalytic cycle. We visualize this previously hypothetical reaction mechanism, directly observing formation of a thioimidate covalent intermediate in ICH microcrystals during catalysis. ICH exhibits a concerted helical displacement upon active-site cysteine modification that is gated by changes in hydrogen bond strength between the cysteine thiolate and the backbone amide of the highly strained Ile152 residue. These catalysis-activated motions permit water entry into the ICH active site for intermediate hydrolysis. Mutations at a Gly residue (Gly150) that modulate helical mobility reduce ICH catalytic turnover and alter its pre-steady-state kinetic behavior, establishing that helical mobility is important for ICH catalytic efficiency. These results demonstrate that MISC can capture otherwise elusive aspects of enzyme mechanism and dynamics in microcrystalline samples, resolving long-standing questions about the connection between nonequilibrium protein motions and enzyme catalysis.

KEYWORDS:

X-ray crystallography; XFEL; cysteine modification; enzyme conformational dynamics; radiation damage

PMID:
31801874
PMCID:
PMC6926069
[Available on 2020-06-04]
DOI:
10.1073/pnas.1901864116

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