A C-TERMINAL CONFORMATIONAL EQUILIBRIUM IN THYMIDYLATE SYNTHASE OBSERVED BY ELECTRON-PARAMAGNETIC-RESONANCE SPECTROSCOPY

Citation
Cw. Carreras et al., A C-TERMINAL CONFORMATIONAL EQUILIBRIUM IN THYMIDYLATE SYNTHASE OBSERVED BY ELECTRON-PARAMAGNETIC-RESONANCE SPECTROSCOPY, Biochemistry, 33(8), 1994, pp. 2071-2077
Citations number
29
Categorie Soggetti
Biology
Journal title
ISSN journal
00062960
Volume
33
Issue
8
Year of publication
1994
Pages
2071 - 2077
Database
ISI
SICI code
0006-2960(1994)33:8<2071:ACCEIT>2.0.ZU;2-6
Abstract
A spin-label was attached to the C-terminal side chain of Lactobacillu s casei thymidylate synthase (TS, EC 2.1.1.45), and EPR spectroscopy w as used to study the change in conformational equilibrium that occurs when the enzyme binds nucleotides or the methylenetetrahydrofolate ana log CB3717. The C244T/V316C mutant TS has only two cysteines, the acti ve site Cys-198 and an engineered cysteine which replaces valine as th e C-terminal residue. dUMP was used to block the active-site cysteine while the C-terminus was reacted with the spin-label 4-maleimido-2,2,6 ,6-tetramethylpiperidinyl-1-oxy. Exclusive attachment of the label to the C-terminal cysteine was verified by a study of the labeled enzyme' s reaction with 5,5'-dithiobis(2-nitrobenzoic acid). EPR spectra of th e labeled enzyme and its complexes were composed of two components cor responding to populations of both flexible and more immobilized forms of the C-terminus (tau(C) = 1 and 9.7 ns, respectively). Ligand bindin g increased the population of the more immobilized form of the C-termi nus with the following series: free enzyme < E.dUMP approximate to dTM P approximate to E.FdUMP < E.CB3717 < E.dUMP.CB3717. Ligand-induced pe rturbation of the conformational equilibrium was titratable and indica ted approximate K-d values of 3 and 13 mu M for formation of the E.dUM P and E.CB3717 binary complexes, respectively, and 7 mu M for the bind ing of CB3717 to the E dUMP complex. Immobilization of the spin-label correlated well with crystallographic B-factors of the C-terminal resi due in corresponding TS crystal structures. These results show that TS has two major conformations which are in equilibrium, and the positio n of the equilibrium changes in the presence of ligands.