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

Beata G. Vertessy, Gunilla Larsson, Tina Persson, Anna Carin Bergman, Rebecca Persson, Per Olof Nyman

Research output: Contribution to journalArticle

42 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)83-88
Number of pages6
JournalFEBS letters
Volume421
Issue number1
DOIs
Publication statusPublished - Jan 2 1998

Keywords

  • Circular dichroism spectroscopy
  • Escherichia coli
  • Flexible C-terminal arm
  • Motif 5
  • Non-hydrolyzable substrate analogue
  • dUTP pyrophosphatase

ASJC Scopus subject areas

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology
  • Genetics
  • Cell Biology

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