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

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, Frank R; 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

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, Frank R; 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.

Afiliação

Smith N; Department of Biochemistry and Redox Biology Center, University of Nebraska-Lincoln, Lincoln, NE, 68588.
Dasgupta M; Department of Biochemistry and Redox Biology Center, University of Nebraska-Lincoln, Lincoln, NE, 68588.
Wych DC; Computer, Computational, and Statistical Sciences Division, Los Alamos National Laboratory, Los Alamos, NM 875405.
Dolamore C; Center for Nonlinear Studies, Los Alamos National Laboratory, Los Alamos, NM 87545.
Sierra RG; Department of Biochemistry and Redox Biology Center, University of Nebraska-Lincoln, Lincoln, NE, 68588.
Lisova S; Linac Coherent Light Source, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, CA 94025.
Marchany-Rivera D; Linac Coherent Light Source, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, CA 94025.
Cohen AE; Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, CA 94025.
Boutet S; Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, CA 94025.
Hunter MS; Linac Coherent Light Source, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, CA 94025.
Kupitz C; Linac Coherent Light Source, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, CA 94025.
Poitevin F; Linac Coherent Light Source, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, CA 94025.
Moss FR; Linac Coherent Light Source, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, CA 94025.
Brewster AS; Linac Coherent Light Source, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, CA 94025.
Sauter NK; Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720.
Young ID; Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720.
Wolff AM; Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720.
Tiwari VK; Department of Chemistry and Biochemistry, University of California, Merced, CA, 93540.
Kumar N; Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE, 68588.
Berkowitz DB; Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE, 68588.
Hadt RG; Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE, 68588.
Thompson MC; Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA USA.
Follmer AH; Department of Chemistry and Biochemistry, University of California, Merced, CA, 93540.
Wall ME; Department of Chemistry, University of California-Irvine, Irvine, CA 92697.
Wilson MA; Computer, Computational, and Statistical Sciences Division, Los Alamos National Laboratory, Los Alamos, NM 875405.

bioRxiv ; 2023 Aug 16.

Article em En | MEDLINE | ID: mdl-37645800

ABSTRACT

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 time-resolved 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.

Palavras-chave

enzyme dynamics; isonitrile; serial crystallography; x-ray free electron laser

Texto completo

Adicionar na Minha BVS

Imprimir

XML

PubMed Links

Buscar no Google