Birdsall D L, Huang W, Santi D V, Stroud R M, Finer-Moore J
Department of Biochemistry and Biophysics, University of California, San Francisco 94143, USA.
Protein Eng. 1998 Mar;11(3):171-83. doi: 10.1093/protein/11.3.171.
X-Ray crystal structures of Lactobacillus casei thymidylate synthase (TS) mutant complexes of E60D with dUMP, and E60Q with dUMP or FdUMP, as well as ternary complexes with folate analog inhibitor CB3717, are described. The structures we report address the decrease in rate of formation of ternary complexes in the E60 mutants. Structures of ternary complexes of L.casei TS mimic ligand-bound TS just prior to covalent bond formation between ligands and protein. Ternary complex structures of L.casei TS E60Q show the ligands are not optimally aligned for making the necessary covalent bonds. Since CB3717 is an analog of the open, activated form of the cofactor, these structures suggest that the slow rate of ternary complex formation in E60 mutants is at least partly the result of impaired alignment of ligands in the active site after binding and activation of the cofactor. Binary complexes of TS E60Q and TS E60D with substrate (dUMP) show no change in dUMP position or occupancy. These results are consistent with the fact that Kd(dUMP) and Km(dUMP) are almost the same, and the rates of folate-independent debromination of 5-bromo-dUMP are even higher than for wild type TS.
本文描述了干酪乳杆菌胸苷酸合成酶（TS）的E60D与dUMP、E60Q与dUMP或FdUMP的突变体复合物，以及与叶酸类似物抑制剂CB3717的三元复合物的X射线晶体结构。我们报道的结构解释了E60突变体中三元复合物形成速率的降低。干酪乳杆菌TS的三元复合物结构模拟了配体与蛋白质之间共价键形成之前的配体结合型TS。干酪乳杆菌TS E60Q的三元复合物结构表明，配体未处于形成必要共价键的最佳排列状态。由于CB3717是辅因子开放、活化形式的类似物，这些结构表明，E60突变体中三元复合物形成速率缓慢至少部分是由于辅因子结合和活化后活性位点中配体排列受损所致。TS E60Q和TS E60D与底物（dUMP）的二元复合物显示dUMP位置或占有率没有变化。这些结果与Kd（dUMP）和Km（dUMP）几乎相同这一事实一致，并且5-溴-dUMP的叶酸非依赖性脱溴速率甚至高于野生型TS。
