Boles J O, Cisneros R J, Weir M S, Odom J D, Villafranca J E, Dunlap R B
Department of Chemistry, University of South Carolina, Columbia 29208.
Biochemistry. 1991 Nov 19;30(46):11073-80. doi: 10.1021/bi00110a009.
Replacement of methionine (Met) residues by selenomethionine (SeMet) was recently shown to facilitate the crystallographic analysis of protein structure through the application of multi-wavelength anomalous diffraction techniques [Yang et al. (1990) Science (Washington, D.C.) 249, 1398-1405]. The availability of SeMet-containing proteins provides an excellent opportunity to evaluate the effects of the complete replacement of Met by SeMet. We chose to compare the properties of selenomethionyl thymidylate synthase isolated from Escherichia coli DL41 (a methionine auxotroph) and wild-type (wt) enzyme obtained from E. coli Rue10. An improved purification procedure for thymidylate synthase was developed which permitted the isolation of 25 mg of pure protein from 2 g of E. coli in 90% yield in no more than 8 h. The pure wt and SeMet enzymes exhibited specific activities 40% higher than published values. Thermal stability studies at 30 degrees C in degassed buffer showed that the SeMet enzyme (t1/2 67 h) was 8-fold less stable than wt enzyme (t1/2 557 h). The half-lives for the latter enzymes in nondegassed buffers at 30 degrees C were decreased by 2-fold, thus indicating the sensitivity of the enzyme to dissolved oxygen. Both enzymes exhibited essentially the same kinetic and binding properties, including Km(dUMP) (1.2 x 10(-6) M), specificity constant (1.6 x 10(6) s-1 M-1), and Kd for 5-fluorodeoxyuridylate binding (1.2 nM) in covalent inhibitory ternary complexes. In addition, X-ray crystallographic analysis by difference Fourier synthesis showed there was no significant difference in conformation between the SeMet enzyme and the wt enzyme.
最近研究表明，通过多波长反常衍射技术的应用，用硒代蛋氨酸（SeMet）取代蛋氨酸（Met）残基有助于蛋白质结构的晶体学分析[Yang等人（1990年），《科学》（华盛顿特区）249卷，第1398 - 1405页]。含SeMet蛋白质的可得性为评估SeMet完全取代Met的影响提供了绝佳机会。我们选择比较从大肠杆菌DL41（蛋氨酸营养缺陷型）分离得到的硒代蛋氨酰胸苷酸合酶与从大肠杆菌Rue10获得的野生型（wt）酶的性质。开发了一种改进的胸苷酸合酶纯化程序，该程序允许在不超过8小时内从2克大肠杆菌中以90%的产率分离出25毫克纯蛋白。纯化后的wt酶和SeMet酶的比活性比已发表的值高40%。在30℃的脱气缓冲液中进行的热稳定性研究表明，SeMet酶（半衰期67小时）的稳定性比wt酶（半衰期557小时）低8倍。后一种酶在30℃的非脱气缓冲液中的半衰期缩短了2倍，这表明该酶对溶解氧敏感。两种酶表现出基本相同的动力学和结合特性，包括共价抑制三元复合物中的Km（dUMP）（1.2×10⁻⁶ M）、特异性常数（1.6×10⁶ s⁻¹ M⁻¹）以及5 - 氟脱氧尿苷酸结合的Kd（1.2 nM）。此外，通过差分傅里叶合成进行的X射线晶体学分析表明，SeMet酶和wt酶在构象上没有显著差异。
