Nakano T, Spencer H T, Appleman J R, Blakley R L
Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101.
Biochemistry. 1994 Aug 23;33(33):9945-52. doi: 10.1021/bi00199a017.
Directed mutagenesis has been used to construct five variants of human dihydrofolate reductase in which smaller residues are substituted for phenylalanine 34, a residue participating in the binding of substrate and methotrexate by interaction with their pteridine rings. The variant enzymes are stable and have decreased affinities for methotrexate (by factors of 2700-60000 at pH 7.65) due to a decreased rate of methotrexate association and a much larger increase in the rate constant for dissociation. However, the catalytic efficiencies of the variants are also lowered by factors of 160-5000, so that it is doubtful whether these enzymes are capable of conferring methotrexate resistance on the cells harboring them. High concentrations of dihydrofolate cause marked inhibition of all the variants, which complicates the determination of kinetic parameters. By the use of stopped-flow spectrophotometry and fluorimetry and other methods, it has been shown that, like the wild-type enzyme, the variants have a branched reaction pathway, but in contrast to the wild-type enzyme, the distribution of flux between alternate pathways is dependent on the concentration of dihydrofolate. This different branch point is a consequence of the very rapid dissociation of tetrahydrofolate from the ternary product complexes of the variant enzymes. Inhibition by dihydrofolate is due to its combination with the enzyme-NADP complex and the slow dissociation of NADP from the resulting abortive complex. When steady state kinetics for this model are simulated using the experimentally determined rate and dissociation constants for the alanine 34 variant, most steady state experimental results are closely approximated.
定向诱变已被用于构建人二氢叶酸还原酶的五个变体，其中用较小的残基取代苯丙氨酸34，该残基通过与底物和甲氨蝶呤的蝶啶环相互作用参与它们的结合。这些变体酶是稳定的，并且对甲氨蝶呤的亲和力降低（在pH 7.65时降低2700 - 60000倍），这是由于甲氨蝶呤结合速率降低以及解离速率常数大幅增加。然而，这些变体的催化效率也降低了160 - 5000倍，因此这些酶是否能够赋予携带它们的细胞甲氨蝶呤抗性值得怀疑。高浓度的二氢叶酸会对所有变体产生显著抑制，这使得动力学参数的测定变得复杂。通过使用停流分光光度法、荧光法和其他方法，已表明与野生型酶一样，这些变体具有分支反应途径，但与野生型酶不同的是，交替途径之间的通量分布取决于二氢叶酸的浓度。这种不同的分支点是变体酶的四氢叶酸从三元产物复合物中非常快速解离的结果。二氢叶酸的抑制作用是由于它与酶 - NADP复合物结合以及NADP从形成的无效复合物中缓慢解离。当使用丙氨酸34变体的实验测定速率和解离常数对该模型的稳态动力学进行模拟时，大多数稳态实验结果都能得到很好的近似。
