Reche P, Arrebola R, Santi D V, Gonzalez-Pacanowska D, Ruiz-Perez L M
Instituto de Parasitología y Biomedicina, Granada, Spain.
Mol Biochem Parasitol. 1996 Feb-Mar;76(1-2):175-85. doi: 10.1016/0166-6851(95)02557-x.
We have cloned and expressed in Escherichia coli a 702-base pair gene coding for the dihydrofolate reductase (DHFR) domain of the bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) from Trypanosoma cruzi. The DHFR domain was purified to homogeneity by methotrexate-Sepharose chromatography followed by an anion-exchange chromatography step in a mono Q column, and displayed a single 27-kDa band on SDS-PAGE. Gel filtration showed that the catalytic domain was expressed as a monomer. Kinetic parameters were similar to those reported for the wild-type bifunctional enzyme with Km values of 0.75 microM for dihydrofolate and 16 microM for NADPH and a kcat value of 16.5 s-1. T. cruzi DHFR is poorly inhibited by trimethoprim and pyrimethamine and the inhibition constants were always lower for the bifunctional enzyme. The binding of methotrexate was characteristic of a class of inhibitors that form an initial complex which isomerizes slowly to a tighter complex and are referred to as 'slow, tight-binding' inhibitors. While the slow-binding step of inhibition was apparently unaffected in the individually expressed DHFR domain, the overall inhibition constant was two-fold higher as a consequence of the superior inhibition constant value obtained for the initial inhibitory complex.
我们已经克隆了来自克氏锥虫的双功能二氢叶酸还原酶-胸苷酸合成酶（DHFR-TS）中编码二氢叶酸还原酶（DHFR）结构域的702个碱基对的基因，并在大肠杆菌中进行了表达。通过甲氨蝶呤-琼脂糖凝胶层析，随后在单Q柱上进行阴离子交换层析步骤，将DHFR结构域纯化至同质，并且在SDS-PAGE上显示出一条单一的27 kDa条带。凝胶过滤显示催化结构域以单体形式表达。动力学参数与野生型双功能酶报道的参数相似，二氢叶酸的Km值为0.75 μM，NADPH的Km值为16 μM，kcat值为16.5 s-1。克氏锥虫DHFR对甲氧苄啶和乙胺嘧啶的抑制作用较弱，并且双功能酶的抑制常数总是更低。甲氨蝶呤的结合具有一类抑制剂的特征，这类抑制剂形成一种初始复合物，该复合物缓慢异构化为一种更紧密的复合物，被称为“缓慢、紧密结合”抑制剂。虽然在单独表达的DHFR结构域中抑制的缓慢结合步骤显然未受影响，但由于初始抑制复合物获得的更高抑制常数，总体抑制常数高出两倍。
