Cody V, Luft J R, Ciszak E, Kalman T I, Freisheim J H
Medical Foundation of Buffalo, NY 14203.
Anticancer Drug Des. 1992 Dec;7(6):483-91.
The crystal structure of the methotrexate-gamma-tetrazole (MTXT)-NADPH ternary complex with recombinant human dihydrofolate reductase (DHFR) has been determined and refined to R = 15.9% for 7003 data from 10.0 to 2.3 A resolution for the R3 lattice. Interpretation of difference Fourier electron density maps revealed that the cofactor NADPH is bound in an extended conformation, and the closest contact between cofactor and inhibitor is 3.1 A, between N(5) of the MTXT pteridine ring and the nicotinamide C(4) which transfers a hydride during the enzyme-catalyzed reaction. As in other DHFR complexes, MTXT is interpreted as protonated at N(1) by Glu-30, and the 2-amino group is hydrogen bonded to a structurally conserved water which also interacts with Glu-30 and Thr-136. The 4-amino group of MTXT hydrogen bonds to the carbonyl of Ile-7 and the phenolic hydroxyl of Tyr-121, and the alpha-carboxylate forms a salt bridge with the conserved Arg-70. In this structure, the amide carbonyl forms two hydrogen bonds with Asn-64 and a water molecule, whereas the gamma-tetrazole ring does not interact directly with the enzyme. The largest changes in the secondary structure on formation of the ternary complex involve the fold of a flexible loop near residues 40-46, and to a lesser extent the helical region near residues 102-109 and the beta-sheet regions near residues 71-75 and 157-159.
已确定甲氨蝶呤 - γ - 四氮唑（MTXT）-NADPH与重组人二氢叶酸还原酶（DHFR）的三元复合物的晶体结构，并针对R3晶格中分辨率为10.0至2.3 Å的7003个数据将其精修至R = 15.9%。对差分傅里叶电子密度图的解读表明，辅因子NADPH以伸展构象结合，辅因子与抑制剂之间的最短接触距离为3.1 Å，位于MTXT蝶啶环的N(5)与烟酰胺C(4)之间，后者在酶催化反应中转移一个氢化物。与其他DHFR复合物一样，MTXT在N(1)处被Glu - 30质子化，其2 - 氨基与一个结构保守的水分子形成氢键，该水分子也与Glu - 30和Thr - 136相互作用。MTXT的4 - 氨基与Ile - 7的羰基和Tyr - 121的酚羟基形成氢键，α - 羧酸盐与保守的Arg - 70形成盐桥。在该结构中，酰胺羰基与Asn - 64和一个水分子形成两个氢键，而γ - 四氮唑环不与酶直接相互作用。三元复合物形成时二级结构的最大变化涉及40 - 46位附近一个柔性环的折叠，以及较小程度上102 - 109位附近的螺旋区域和71 - 75位及157 - 159位附近的β - 折叠区域。
