ISOMORPHOUS CRYSTAL-STRUCTURES OF ESCHERICHIA-COLI DIHYDROFOLATE-REDUCTASE COMPLEXED WITH FOLATE, 5-DEAZAFOLATE, AND 5,10-DIDEAZATETRAHYDROFOLATE - MECHANISTIC IMPLICATIONS
ISOMORPHOUS CRYSTAL-STRUCTURES OF ESCHERICHIA-COLI DIHYDROFOLATE-REDUCTASE COMPLEXED WITH FOLATE, 5-DEAZAFOLATE, AND 5,10-DIDEAZATETRAHYDROFOLATE - MECHANISTIC IMPLICATIONS
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DOI：
10.1021/bi00008a039
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发表时间：
1995-02-28
期刊：
影响因子：
2.9
通讯作者：
KRAUT, J
中科院分区：
文献类型：
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作者：
REYES, VM;SAWAYA, MR;KRAUT, J
在1.9埃时，大肠杆菌二氢叶酸还原酶(ecDHFR，EC 1.5.1.3)与叶酸、5-去氮卓酮(5dfol)和5，10-二氮杂四氢叶酸(DdTHF)形成的二元络合物的晶体结构分别被细化到13.7%、14.9%和14.5%。这三种化合物都与先前报道的ecDHFR与甲氨蝶呤(MTX)的二元配合物同构，在P6(1)空间群中，每个不对称单元有两个分子[Bolin，J.T.，Filman，D.J.，Matthews，D.A.，Hamlin，R.C.，&Kraut，J.(1982)J.Biol]。化学。257、13650-13662]。到目前为止还没有观察到的水分子与叶酸络合物(但不是5dfol或ddTHF络合物)不对称单元的两个分子中的蝶啶N5和O4氢键，支持了结合底物的N5质子化是DHFR反应的重要步骤通过这样的水分子发生的假设。没有迹象表明5dfol的N8与Ile-5的主链羰基之间存在氢键，这表明细菌酶不同于人类的酶[Davies，J.F.，II，DelCamp，T.J.，Prendergast，N.J.，Ashford，V.A.，Freisheim，J.H.，&Kraut，J.(1990)BioChemical 29,9467-9479]，也不利于N8的质子化。也许这就解释了为什么细菌DHFR在减少叶酸方面远不如脊椎动物DHFR有效。当ecDHFR。NADPH络合物(空间群P3(2)21；M.R.Sawaya，正在制备中)被重叠在叶酸和5dfol络合物上，从蝶啶C6到烟酰胺C4的距离分别为2.9埃和2.8埃，与理论计算的氢化物转移过渡态的最佳距离非常一致[Wu，Y.D.，&Houk，K.N.(1987)J.Am化学。SoC。109、906-908、2226-2227]。与叶酸或5dfol的平面环系不同，ddTHF的还原的蝶烷环严重皱折并向烟酰胺口袋弯曲，还原的吡啶环呈半椅型构象。这种形状的变化导致蝶啶环与O4更接近Trp-22(N Epsilon 1)超过0.5埃，因此不变的水分子现在以理想的氢键连接这两个原子。此外，虽然叶酸和5dfol的PABA环几乎重合，并且比αB螺旋更接近αC螺旋，但MTX和ddTHF的PABA环沿结合缝隙分别位移约1.1和0.6埃，与αB和αC等距离。这两个特征可以解释THF从酶-产物复合体中缓慢释放的原因。最后，当NADPH络合物与ddTHF络合物重叠时，我们发现蝶啶C7和烟酰胺C4之间有完整的2.0 Angstrom van der Waals重叠，这可能解释了辅因子结合加速THF释放的原因。
Crystal structures of Escherichia coli dihydrofolate reductase (ecDHFR, EC 1.5.1.3) in binary complexes with folate, 5-deazafolate (5dfol), and 5,10-dideazatetrahydrofolate (ddTHF) have been refined to R-factors of 13.7%, 14.9%, and 14.5%, respectively, all at 1.9 Angstrom. All three are isomorphous with a previously reported binary complex of ecDHFR with methotrexate (MTX), in space group P6(1), two molecules per asymmetric unit [Bolin, J. T., Filman, D. J., Matthews, D. A., Hamlin, R. C., & Kraut, J. (1982) J. Biol. Chem. 257, 13650-13662]. A hitherto unobserved water molecule is hydrogen bonded to the pteridine N5 and O4 in both molecules of the asymmetric unit of the folate complex (but not the 5dfol or ddTHF complexes), supporting the hypothesis that N5 protonation of bound substrate, an important step of the DHFR reaction, occurs by way of such a water molecule. There is no indication of a hydrogen bond between N8 of 5dfol and the backbone carbonyl of Ile-5, suggesting that the bacterial enzyme, unlike the human enzyme [Davies, J. F., II, Delcamp, T. J., Prendergast, N. J., Ashford, V. A., Freisheim, J. H., & Kraut, J. (1990) Biochemistry 29, 9467-9479], does nor favor protonation at N8. Perhaps this explains why bacterial DHFR is much less effective than vertebrate DHFR in folate reduction. When the ecDHFR . NADPH complex (space group P3(2)21; M. R. Sawaya, in preparation) is superimposed on the folate and 5dfol complexes, the distances from pteridine C6 to nicotinamide C4 were found to be 2.9 and 2.8 Angstrom, respectively, in close agreement with the theoretically calculated optimal distance in the transition state for hydride transfer [Wu, Y. D., & Houk, K. N. (1987) J. Am. Chem. Soc. 109, 906-908, 2226-2227]. In contrast to the planar ring system of folate or 5dfol, the reduced pteridine ring of ddTHF is severely puckered and bent toward the nicotinamide pocket, with the reduced pyridine ring assuming a half-chair type of conformation. This change in shape causes the pteridine ring to bind with O4 closer to Trp-22(N epsilon 1) by over 0.5 Angstrom, so that an invariant water molecule now bridges these two atoms with ideal hydrogen bonds. Furthermore, while the pABA rings of folate and 5dfol are nearly coincident and closer to the alpha C helix than to the alpha B helix, those of MTX and ddTHF are displaced along the binding crevice by approximately 1.1 and 0.6 Angstrom, respectively, and are equidistant from alpha B and alpha C. These two features could explain the slow release of THF from the enzyme-product complex. Finally, when the NADPH complex is superimposed with the ddTHF complex, we find a full 2.0 Angstrom van der Waals overlap between the pteridine C7 and nicotinamide C4, perhaps explaining the acceleration of THF release by cofactor binding.