Thillet J, Absil J, Stone S R, Pictet R
Unité 257 de l'Institut National de la Santé et de la Recherche Médicale, Institut Jacques Monod du Centre National de la Recherche Scientifique, Paris, France.
J Biol Chem. 1988 Sep 5;263(25):12500-8.
Site-directed mutagenesis was used to generate mutants of recombinant mouse dihydrofolate reductase to test the role of some amino acids in the binding of two inhibitors, methotrexate and trimethoprim. Eleven mutations changing eight amino acids at positions all involved in hydrogen bonding or hydrophobic interactions with dihydrofolate or one of the two inhibitors were tested. Nine mutants were obtained by site-directed mutagenesis and two were spontaneous mutants previously obtained by in vivo selection (Grange, T., Kunst, F., Thillet, J., Ribadeau-Dumas, B., Mousseron, S., Hung, A., Jami, J., and Pictet, R. (1984) Nucleic Acids Res. 12, 3585-3601). The choice of the mutated positions was based on the knowledge of the active site of chicken dihydrofolate reductase established by x-ray crystallographic studies since the sequences of all known eucaryotic dihydrofolate reductases are greatly conserved. Enzymes were produced in great amounts and purified using a plasmid expressing the mouse cDNA into a dihydrofolate reductase-deficient Escherichia coli strain. The functional properties of recombinant mouse dihydrofolate reductase purified from bacterial extracts were identical to those of dihydrofolate reductase isolated from eucaryotic cells. The Km(NADPH) values for all the mutants except one (Leu-22----Arg) were only slightly modified, suggesting that the mutations had only minor effects on the ternary conformation of the enzyme. In contrast, all Km(H2folate) values were increased, since the mutations were located in the dihydrofolate binding site. The catalytic activity was also modified for five mutants with, respectively, a 6-, 10-, 36-, and 60-fold decrease of Vmax for Phe-31----Arg, Ile-7----Ser, Trp 24----Arg and Leu-22----Arg mutants and a 2-fold increase for Val-115----Pro. All the mutations affected the binding of methotrexate and six, the binding of trimethoprim: Ile-7----Ser, Leu-22----Arg, Trp-24----Arg, Phe-31----Arg, Gln-35----Pro and Phe-34----Leu. The relative variation of Ki for methotrexate and trimethoprim were not comparable from one mutant to the next, reflecting the different binding modes of the two inhibitors. The mutations which yielded the greatest increases in Ki are those which involved amino acids making hydrophobic contacts with the inhibitor.
采用定点诱变技术构建重组小鼠二氢叶酸还原酶突变体，以检测某些氨基酸在两种抑制剂甲氨蝶呤和甲氧苄啶结合中的作用。测试了11个突变，这些突变改变了8个氨基酸位点，这些位点均参与与二氢叶酸或两种抑制剂之一的氢键或疏水相互作用。通过定点诱变获得了9个突变体，另外2个是先前通过体内筛选获得的自发突变体（格兰奇，T.，昆斯特，F.，蒂耶，J.，里巴多 - 迪马斯，B.，穆瑟龙，S.，洪，A.，贾米，J.，皮克泰，R.（1984年）《核酸研究》12，3585 - 3601）。突变位点的选择基于通过X射线晶体学研究确定的鸡二氢叶酸还原酶活性位点的知识，因为所有已知真核生物二氢叶酸还原酶的序列高度保守。使用将小鼠cDNA表达为二氢叶酸还原酶缺陷型大肠杆菌菌株的质粒大量生产并纯化酶。从细菌提取物中纯化的重组小鼠二氢叶酸还原酶的功能特性与从真核细胞中分离的二氢叶酸还原酶相同。除一个突变体（Leu - 22→Arg）外，所有突变体的Km（NADPH）值仅略有改变，这表明这些突变对酶的三元构象影响较小。相反，所有的Km（H2叶酸）值都增加了，因为这些突变位于二氢叶酸结合位点。五个突变体的催化活性也发生了改变，Phe - 31→Arg、Ile - 7→Ser、Trp 24→Arg和Leu - 22→Arg突变体的Vmax分别降低了6倍、10倍、36倍和60倍，而Val - 115→Pro突变体的Vmax增加了2倍。所有突变均影响甲氨蝶呤的结合，六个突变影响甲氧苄啶的结合：Ile - 7→Ser、Leu - 22→Arg、Trp - 24→Arg、Phe - 31→Arg、Gln - 35→Pro和Phe - 34→Leu。从一个突变体到另一个突变体，甲氨蝶呤和甲氧苄啶的Ki相对变化不可比，这反映了两种抑制剂不同的结合模式。导致Ki增加最大的突变是那些涉及与抑制剂形成疏水接触的氨基酸的突变。
