Fantz C, Shaw D, Jennings W, Forsthoefel A, Kitchens M, Phan J, Minor W, Lebioda L, Berger F G, Spencer H T
Department of Chemistry, Columbia, South Carolina, USA.
Mol Pharmacol. 2000 Feb;57(2):359-66.
Drug-resistant variants of thymidylate synthase (TS) can potentially be used in gene therapy applications to decrease the myelosuppressive side effects of TS-directed anticancer agents or to select genetically modified cells in vivo. Mutations of proline 303 of human TS confer resistance to TS-directed fluoropyrimidines and antifolates (). We generated the corresponding variants in Escherichia coli TS (ecTS), position 254, to better understand the mechanism by which mutations at this residue confer resistance. In addition, because ecTS is intrinsically resistant to several antifolates when compared with human TS, we suspected that greater resistance could be achieved with the bacterial enzyme. The P254L enzyme conferred >100-fold resistance to both raltitrexed and 5-fluoro-2'-deoxyuridine (FdUrd) compared with wild-type ecTS. Four additional mutants (P254F, P254S, P254G, and P254D), each of which complemented growth of a TS-deficient cell line, were generated, isolated, and characterized. Steady-state values of K(m) for dUMP and k(cat) were not substantially different among the variants and were comparable with the wild-type values, but K(m) for methylenetetrahydrofolate (CH(2)H(4)PteGlu) was >10-fold higher for P254D. Values of k(on) and k(off) for nucleotide binding, which were obtained by stopped-flow spectroscopy, were virtually unchanged among the mutants. Drastic differences were observed for CH(2)H(4)PteGlu binding, with K(d) values >15-fold higher than observed with the wild-type enzyme; surprisingly, the proposed isomerization reaction that is very evident for the wild-type enzyme is not observed with P254S. The decrease in affinity for CH(2)H(4)PteGlu correlates well with K(i) values obtained for three TS-directed inhibitors. These results show that mutations at Pro-254 specifically affect the initial binding interactions between enzyme and cofactor and also alter the ability of the mutant enzymes to undergo conformational changes that occur on ternary complex formation. The crystal structure of P254S was determined at 1.5 A resolution and is the most precise structure of TS available. When compared with wild-type TS, the structure shows local conformational changes affecting mostly Asp-253; its carbonyl is rotated approximately 40 degrees, and the side chain forms an ion pair with Arg-225.
胸苷酸合成酶（TS）的耐药变体可潜在地用于基因治疗，以降低TS导向抗癌药物的骨髓抑制副作用，或在体内选择基因修饰细胞。人TS脯氨酸303位点的突变赋予对TS导向氟嘧啶和抗叶酸剂的抗性（）。我们在大肠杆菌TS（ecTS）的相应位置254生成了相应变体，以更好地理解该残基处的突变赋予抗性的机制。此外，由于与人类TS相比，ecTS对几种抗叶酸剂具有内在抗性，我们怀疑细菌酶可能具有更高的抗性。与野生型ecTS相比，P254L酶对雷替曲塞和5-氟-2'-脱氧尿苷（FdUrd）的抗性均提高了100倍以上。我们生成、分离并表征了另外四个突变体（P254F、P254S、P254G和P254D），每个突变体都能补充TS缺陷细胞系的生长。各变体中dUMP的K(m)稳态值和k(cat)没有显著差异，与野生型值相当，但P254D的亚甲基四氢叶酸（CH(2)H(4)PteGlu）的K(m)值高出10倍以上。通过停流光谱法获得的核苷酸结合的k(on)和k(off)值在突变体之间几乎没有变化。观察到CH(2)H(4)PteGlu结合存在显著差异，其K(d)值比野生型酶高出15倍以上；令人惊讶的是，野生型酶中非常明显的拟异构化反应在P254S中未观察到。对CH(2)H(4)PteGlu亲和力的降低与三种TS导向抑制剂的K(i)值密切相关。这些结果表明，脯氨酸254位点的突变特异性地影响酶与辅因子之间的初始结合相互作用，也改变了突变酶在三元复合物形成时发生构象变化的能力。P254S的晶体结构在1.5埃分辨率下确定，是现有最精确的TS结构。与野生型TS相比，该结构显示出主要影响天冬氨酸253的局部构象变化；其羰基旋转约40度，侧链与精氨酸225形成离子对。
