| 1. |
- Vertessy, Beata G, et al.
(författare)
-
Specific Derivatization of the Active Site Tyrosine in dUTPase Perturbs Ligand Binding to the Active Site
- 1996
-
Ingår i: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 1090-2104 .- 0006-291X. ; 219:2, s. 294-300
-
Tidskriftsartikel (refereegranskat)abstract
- Selective modification of one (of three) tyrosine residue per enzyme monomer leads to inactivation of dUTPase of the retrovirus equine infectious anemia virus (EIAV). The substrate dUMP and the cofactor Mg2+protects against inactivation and modification, in agreement with the study onE. colidUTPase (Vertessyet al.(1994)Biochim. Biophys. Acta1205, 146-150). Amino acid analyses of nitrated dUTPases confirmed Tyr-selectivity of modification. The nitrated residue inE. colidUTPase was identified as the evolutionary highly conserved Tyr-93. The modifiable residue is shown to be the only Tyr exposed in bothE. coliand EIAV dUTPases. As a consequence of Tyr-93 derivatization, the Mg2+-dependent interaction between the substrate-analogue dUDP andE. colidUTPase becomes impaired as shown by circular dichroism spectroscopy, here presented as a tool for monitoring ligand binding to the active site.
|
|
| 2. |
- Vertessy, Beata G., et al.
(författare)
-
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
-
Ingår i: FEBS Letters. - 1873-3468. ; 421:1, s. 83-88
-
Tidskriftsartikel (refereegranskat)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.
|
|
