Kinetics of the formation and isomerization of methotrexate complexes of recombinant human dihydrofolate reductase.
Kinetics of the formation and isomerization of methotrexate complexes of recombinant human dihydrofolate reductase.
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发表时间：
1988-07
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通讯作者：
J. Appleman;N. Prendergast;T. Delcamp;J. Freisheim;Raymond L. Blakley
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作者：
J. Appleman;N. Prendergast;T. Delcamp;J. Freisheim;Raymond L. Blakley
研究了抑制剂与高纯度重组人二氢叶酸还原酶（rHDHFR）结合的动力学。甲氨蝶呤（MTX）可快速（kon = 1.0 × 10（8）M-1 s-1）和紧密（koff/kon = 210 pM）结合rHDHFR与NADPH的预形成复合物。rHDHFR、NADPH和MTX之间的初始缔合反应之后是所得到的复合物的异构化（Ki = 0.4 s-1），导致新的构象异构体，其中MTX结合得更紧密（Ki = 3.4 pM）。MTX的主要代谢产物具有四个额外的谷氨酸残基，Ki = 1.4 pM，也得到了类似的结果。MTX的另一种主要代谢物7-羟基MTX是rHDHFR的弱抑制剂（Ki = 8.9 nM），该代谢物的聚谷氨酸盐形式也是同样弱的抑制剂（Ki = 9.9 nM），因此向MTX或7-羟基MTX中添加谷氨酸盐残基对其结合几乎没有影响。因此，MTX聚谷氨酸盐形成的意义与其他作用有关，例如通过延长药物的细胞内滞留来增加MTX的细胞毒性。另一种抗叶酸剂甲氧苄啶与细菌来源的二氢叶酸还原酶紧密结合，但与三元复合物中的rHDHFR结合较弱（KD = 0.5 μ M）。虽然缔合步骤是快速的（kon = 0.4 × 10（8）M-1 s-1），但解离速率也是快速的（koff = 15 s-1）。此外，与甲氧苄啶与细菌二氢叶酸还原酶的复合物的已知异构化相反，甲氧苄啶与rHDHFR的三元复合物没有异构化。
The kinetics of inhibitor binding to highly purified recombinant human dihydrofolate reductase (rHDHFR) have been examined. Methotrexate (MTX) binds rapidly (kon = 1.0 x 10(8) M-1 s-1) and tightly (koff/kon = 210 pM) to the preformed complex of rHDHFR with NADPH. The initial association reaction between rHDHFR.NADPH and MTX is followed by an isomerization of the resulting complex (kiso = 0.4 s-1) leading to a new conformer in which MTX is bound even more tightly (Ki = 3.4 pM). Similar results have been obtained with a major metabolite of MTX having four additional glutamate residues for which Ki = 1.4 pM. 7-HydroxyMTX, another major metabolite of MTX, is a weak inhibitor of rHDHFR (Ki = 8.9 nM), and a polyglutamate form of this metabolite is an equally weak inhibitor (Ki = 9.9 nM), so that the addition of glutamate residues to MTX or 7-hydroxyMTX has little effect on their binding. It follows that the significance of MTX polyglutamate formation relates to other roles such as increasing the cytotoxicity of MTX by prolonging intracellular retention of the drug. Another antifolate, trimethoprim, binds tightly to dihydrofolate reductases from bacterial sources, but weakly to rHDHFR in the ternary complex (KD = 0.5 microM). Although the association step is rapid (kon = 0.4 x 10(8) M-1 s-1), the dissociation rate is also rapid (koff = 15 s-1). Furthermore, there is no isomerization of the ternary complex of trimethoprim with rHDHFR, in contrast to the known isomerization of complexes of trimethoprim with bacterial dihydrofolate reductases.