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
(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)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/125625
- author
- Vertessy, Beata G. ; Larsson, Gunilla ; Persson, Tina ; Bergman, Anna-Carin ; Persson, Rebecca and Nyman, Per-Olof LU
- organization
- publishing date
- 1998
- 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
- 2016-04-01 17:12:27
- date last changed
- 2022-01-29 01:06:21
@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}},
keywords = {{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}},
doi = {{10.1016/S0014-5793(97)01545-7}},
volume = {{421}},
year = {{1998}},
}