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The complete triphosphate moiety of non-hydrolyzable substrate analogues is required for a conformational shift of the flexible C-terminus in E. coli dUTP pyrophosphatase

Vertessy, Beata G.; Larsson, Gunilla; Persson, Tina; Bergman, Anna-Carin; Persson, Rebecca and Nyman, Per-Olof LU (1998) In FEBS Letters 421(1). p.83-88
Abstract
The molecular mechanism of substrate analogue interaction with Escherichia coli dUTPase was investigated, using the non-hydrolyzable 2'-deoxyuridine 5'-(α,β-imido)triphosphate (α,β-imido-dUTP). Binding of this analogue induces a difference in the far UV circular dichroism (CD) spectrum arguing for a significant change in protein conformation. The spectral shift is strictly Mg2+-dependent, does not appear with dUDP instead of α,β-imido-dUTP and is not elicited if the flexible C-terminal arm is deleted from the protein by limited tryptic digestion. Involvement of the C-terminal arm in α,β-imido-dUTP binding is consistent with the finding that this analogue protects against tryptic hydrolysis at Arg-141. Near UV CD of ligand-enzyme complexes... (More)
The molecular mechanism of substrate analogue interaction with Escherichia coli dUTPase was investigated, using the non-hydrolyzable 2'-deoxyuridine 5'-(α,β-imido)triphosphate (α,β-imido-dUTP). Binding of this analogue induces a difference in the far UV circular dichroism (CD) spectrum arguing for a significant change in protein conformation. The spectral shift is strictly Mg2+-dependent, does not appear with dUDP instead of α,β-imido-dUTP and is not elicited if the flexible C-terminal arm is deleted from the protein by limited tryptic digestion. Involvement of the C-terminal arm in α,β-imido-dUTP binding is consistent with the finding that this analogue protects against tryptic hydrolysis at Arg-141. Near UV CD of ligand-enzyme complexes reveals a characteristic difference in the microenvironments of enzyme-bound dUDP and α,β-imido-dUTP, a difference not observable in C-terminally truncated dUTPase. The results suggest that (i) closing of the active site during the catalytic cycle, through the movement of the C-terminal arm, requires the presence of the complete triphosphate moiety of the substrate in complex with Mg2+, and (ii) after catalytic cleavage the active site pops open to facilitate product release. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
dUTP pyrophosphatase, Non-hydrolyzable substrate analogue, Circular dichroism spectroscopy, Flexible C-terminal arm, Motif 5, Escherichia coli
in
FEBS Letters
volume
421
issue
1
pages
83 - 88
publisher
Wiley-Blackwell
external identifiers
  • scopus:0032472214
ISSN
1873-3468
DOI
10.1016/S0014-5793(97)01545-7
language
English
LU publication?
yes
id
bcdca115-e16a-48d6-89bc-b9d8201f2ec1 (old id 125625)
date added to LUP
2007-07-09 09:16:36
date last changed
2017-01-08 05:19:38
@article{bcdca115-e16a-48d6-89bc-b9d8201f2ec1,
  abstract     = {The molecular mechanism of substrate analogue interaction with Escherichia coli dUTPase was investigated, using the non-hydrolyzable 2'-deoxyuridine 5'-(α,β-imido)triphosphate (α,β-imido-dUTP). Binding of this analogue induces a difference in the far UV circular dichroism (CD) spectrum arguing for a significant change in protein conformation. The spectral shift is strictly Mg2+-dependent, does not appear with dUDP instead of α,β-imido-dUTP and is not elicited if the flexible C-terminal arm is deleted from the protein by limited tryptic digestion. Involvement of the C-terminal arm in α,β-imido-dUTP binding is consistent with the finding that this analogue protects against tryptic hydrolysis at Arg-141. Near UV CD of ligand-enzyme complexes reveals a characteristic difference in the microenvironments of enzyme-bound dUDP and α,β-imido-dUTP, a difference not observable in C-terminally truncated dUTPase. The results suggest that (i) closing of the active site during the catalytic cycle, through the movement of the C-terminal arm, requires the presence of the complete triphosphate moiety of the substrate in complex with Mg2+, and (ii) after catalytic cleavage the active site pops open to facilitate product release.},
  author       = {Vertessy, Beata G. and Larsson, Gunilla and Persson, Tina and Bergman, Anna-Carin and Persson, Rebecca and Nyman, Per-Olof},
  issn         = {1873-3468},
  keyword      = {dUTP pyrophosphatase,Non-hydrolyzable substrate analogue,Circular dichroism spectroscopy,Flexible C-terminal arm,Motif 5,Escherichia coli},
  language     = {eng},
  number       = {1},
  pages        = {83--88},
  publisher    = {Wiley-Blackwell},
  series       = {FEBS Letters},
  title        = {The complete triphosphate moiety of non-hydrolyzable substrate analogues is required for a conformational shift of the flexible C-terminus in E. coli dUTP pyrophosphatase},
  url          = {http://dx.doi.org/10.1016/S0014-5793(97)01545-7},
  volume       = {421},
  year         = {1998},
}