Carreras C W, Naber N, Cooke R, Santi D V
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143-0448.
Biochemistry. 1994 Mar 1;33(8):2071-7. doi: 10.1021/bi00174a013.
A spin-label was attached to the C-terminal side chain of Lactobacillus casei thymidylate synthase (TS, EC2.1.1.45), and EPR spectroscopy was used to study the change in conformational equilibrium that occurs when the enzyme binds nucleotides or the methylenetetrahydrofolate analog CB3717. The C244T/V316C mutant TS has only two cysteines, the active site Cys-198 and an engineered cysteine which replaces valine as the 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 the labeled enzyme and its complexes were composed of two components corresponding to populations of both flexible and more immobilized forms of the C-terminus (tau C = 1 and 9.7 ns, respectively). Ligand binding increased the population of the more immobilized form of the C-terminus with the following series: free enzyme < E.dUMP approximately dTMP approximately E.FdUMP < E.CB3717 < E.dUMP.CB3717. Ligand-induced perturbation of the conformational equilibrium was titratable and indicated approximate Kd values of 3 and 13 microM for formation of the E.dUMP and E.CB3717 binary complexes, respectively, and 7 microM for the binding of CB3717 to the E.dUMP complex. Immobilization of the spin-label correlated well with crystallographic B-factors of the C-terminal residue in corresponding TS crystal structures. These results show that TS has two major conformations which are in equilibrium, and the position of the equilibrium changes in the presence of ligands.
将一个自旋标记连接到干酪乳杆菌胸苷酸合成酶（TS，EC2.1.1.45）的C末端侧链上，并用电子顺磁共振光谱研究该酶结合核苷酸或亚甲基四氢叶酸类似物CB3717时发生的构象平衡变化。C244T/V316C突变体TS只有两个半胱氨酸，即活性位点的Cys-198和一个经工程改造的半胱氨酸，该半胱氨酸取代缬氨酸作为C末端残基。使用dUMP封闭活性位点的半胱氨酸，同时使C末端与自旋标记4-马来酰亚胺-2,2,6,6-四甲基哌啶基-1-氧基反应。通过研究标记酶与5,5'-二硫代双（2-硝基苯甲酸）的反应，证实了标记仅特异性地连接到C末端半胱氨酸上。标记酶及其复合物的电子顺磁共振光谱由两个成分组成，分别对应于C末端的柔性形式和更固定形式的群体（τC分别为1和9.7纳秒）。配体结合增加了C末端更固定形式的群体，顺序如下：游离酶＜E.dUMP≈dTMP≈E.FdUMP＜E.CB3717＜E.dUMP.CB3717。配体诱导的构象平衡扰动是可滴定的，表明形成E.dUMP和E.CB3717二元复合物的近似解离常数（Kd）值分别为3和13微摩尔，而CB3717与E.dUMP复合物结合的Kd值为7微摩尔。自旋标记的固定与相应TS晶体结构中C末端残基的晶体学B因子密切相关。这些结果表明，TS有两种处于平衡状态的主要构象，并且在配体存在时平衡位置会发生变化。
