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Catalytic cycle of human glutathione reductase near 1 Å resolution

(2008) JOURNAL OF MOLECULAR BIOLOGY. 382(2). p.371-384
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Abstract
Efficient enzyme catalysis depends on exquisite details of structure beyond those resolvable in typical medium- and high-resolution crystallographic analyses. Here we report synchrotron-based cryocrystallographic studies of natural substrate complexes of the flavoenzyme human glutathione, reductase (GR) at nominal resolutions between 1.1 and 0.95 angstrom that reveal new aspects of its mechanism. Compression in the active site causes overlapping van der Waals radii and distortion in the nicotinamide ring of the NADPH substrate, which enhances catalysis via stereoelectronic effects. The bound NADPH and redox-active disulfide are positioned optimally on opposite sides of the flavin for a 1,2-addition across a flavin double bond. The new structures extend earlier observations to reveal that the redox-active disulfide loop in GR is an extreme case of sequential peptide bonds systematically deviating from planarity - a net deviation of 53 degrees across five residues. But this apparent strain is not a factor in catalysis, as it is present in both oxidized and reduced structures. Intriguingly, the flavin bond lengths in oxidized GR are intermediate between those expected for oxidized and reduced flavin, but we present evidence that this may not be clue to the protein environment but instead due to partial synchrotron reduction of the flavin by the synchrotron beam. Finally of more general relevance, we present evidence that the structures of synchrotron-reduced disulfide bonds cannot generally be used as reliable models for naturally reduced disulfide bonds. (C) 2008 Elsevier Ltd. All rights reserved.
Keywords
steric compression, enzyme mechanism, radiation damage, peptide planarity, stereoelectronic control

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Chicago
Berkholzl, Donald, H. Richard Faber, Savvas Savvides, and P. Andrew Karplus. 2008. “Catalytic Cycle of Human Glutathione Reductase Near 1 Å Resolution.” Journal of Molecular Biology 382 (2): 371–384.
APA
Berkholzl, D., Faber, H. R., Savvides, S., & Karplus, P. A. (2008). Catalytic cycle of human glutathione reductase near 1 Å resolution. JOURNAL OF MOLECULAR BIOLOGY, 382(2), 371–384.
Vancouver
1.
Berkholzl D, Faber HR, Savvides S, Karplus PA. Catalytic cycle of human glutathione reductase near 1 Å resolution. JOURNAL OF MOLECULAR BIOLOGY. LONDON NW1 7DX, ENGLAND: ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD; 2008;382(2):371–84.
MLA
Berkholzl, Donald, H. Richard Faber, Savvas Savvides, et al. “Catalytic Cycle of Human Glutathione Reductase Near 1 Å Resolution.” JOURNAL OF MOLECULAR BIOLOGY 382.2 (2008): 371–384. Print.
@article{662540,
  abstract     = {Efficient enzyme catalysis depends on exquisite details of structure beyond those resolvable in typical medium- and high-resolution crystallographic analyses. Here we report synchrotron-based cryocrystallographic studies of natural substrate complexes of the flavoenzyme human glutathione, reductase (GR) at nominal resolutions between 1.1 and 0.95 angstrom that reveal new aspects of its mechanism. Compression in the active site causes overlapping van der Waals radii and distortion in the nicotinamide ring of the NADPH substrate, which enhances catalysis via stereoelectronic effects. The bound NADPH and redox-active disulfide are positioned optimally on opposite sides of the flavin for a 1,2-addition across a flavin double bond. The new structures extend earlier observations to reveal that the redox-active disulfide loop in GR is an extreme case of sequential peptide bonds systematically deviating from planarity - a net deviation of 53 degrees across five residues. But this apparent strain is not a factor in catalysis, as it is present in both oxidized and reduced structures. Intriguingly, the flavin bond lengths in oxidized GR are intermediate between those expected for oxidized and reduced flavin, but we present evidence that this may not be clue to the protein environment but instead due to partial synchrotron reduction of the flavin by the synchrotron beam. Finally of more general relevance, we present evidence that the structures of synchrotron-reduced disulfide bonds cannot generally be used as reliable models for naturally reduced disulfide bonds. (C) 2008 Elsevier Ltd. All rights reserved.},
  author       = {Berkholzl, Donald and Faber, H. Richard and Savvides, Savvas and Karplus, P. Andrew},
  issn         = {0022-2836},
  journal      = {JOURNAL OF MOLECULAR BIOLOGY},
  keyword      = {steric compression,enzyme mechanism,radiation damage,peptide planarity,stereoelectronic control},
  language     = {eng},
  number       = {2},
  pages        = {371--384},
  publisher    = {ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD},
  title        = {Catalytic cycle of human glutathione reductase near 1 {\AA} resolution},
  url          = {http://dx.doi.org/10.1016/j.jmb.2008.06.083},
  volume       = {382},
  year         = {2008},
}

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