DIHYDROFOLATE-REDUCTASE - MULTIPLE CONFORMATIONS AND ALTERNATIVE MODES OF SUBSTRATE BINDING
DIHYDROFOLATE-REDUCTASE - MULTIPLE CONFORMATIONS AND ALTERNATIVE MODES OF SUBSTRATE BINDING
复制标题
DOI：
10.1021/bi00431a048
复制
发表时间：
1989-03-07
期刊：
影响因子：
2.9
通讯作者：
ROBERTS, GCK
中科院分区：
文献类型：
--
作者：
BIRDSALL, B;FEENEY, J;ROBERTS, GCK
干酪乳杆菌二氢叶酸还原酶与底物叶酸和辅酶NADP+的复合物已显示作为三种缓慢相互转化构象的混合物存在于溶液中，所述构象的比例依赖于pH [Birdsall，B.，Gronenborn，A. M.，Hyde，E.一、克洛尔，G。M.，罗伯茨，G。C. K.，Feeney，J.，[Burgen，A.S.V.（1982）Biochemistry 21，5831]。的所有芳香族质子的配体分子的共振在所有三个构象状态的配合物的分配现在已经完成了通过使用各种NMR方法，特别是二维交换实验。辅酶的烟酰胺质子和叶酸的蝶啶7-质子的共振在三种构象中具有不同的化学位移，在某些情况下相差超过1 ppm。在低pH值（构象I）和高pH值（构象IIa和IIb）与酶-甲氨蝶呤-NADP+复合物的COSY光谱的比较表明，在蛋白质的构象只有轻微的差异。配体和蛋白质的化学位移变化模式表明，结构差异定位在酶的活性位点内。在低pH和高pH下，在烟酰胺5-和6-质子与Thr 45的甲基共振之间观察到核奥弗豪泽效应（NOE），表明烟酰胺环没有大的运动。相比之下，在构象I和IIa中，在蝶啶7-质子与Leu 19和Leu 27的甲基质子之间观察到NOE，但在构象IIb中没有。提出了一个模型，可以定性地解释所观察到的NOE和配体1H化学位移。在该模型中，构象I和IIa中的叶酸蝶啶环的结合方式与酶-甲氨蝶呤-NADPH复合物晶体结构中甲氨蝶呤蝶啶环的结合方式相似。然而，在构象IIb中，它在结合位点具有完全不同的取向，与状态I和IIa中的取向相关，旋转约180 °。绕着大致沿着C2·sbd·NH 2键的轴。后一种取向可以解释所观察到的酶还原叶酸的立体化学。因此，叶酸能够以生产性和完全不同的非生产性模式与酶结合，而抑制剂甲氨蝶呤仅以非生产性模式结合。
The complex of Lactobacillus casei dihydrofolate reductase with the substrate folate and the coenzyme NADP+ has been shown to exist in solution as a mixture of three slowly interconverting conformations whose proportions are pH-dependent [Birdsall, B., Gronenborn, A. M., Hyde, E. I., Clore, G. M., Roberts, G. C. K., Feeney, J., [Burgen, A.S.V. (1982) Biochemistry 21, 5831]. The assignment of the resonances of all the aromatic protons of the ligand molecules in all three conformational states of the complex has now been completed by using a variety of NMR methods, particularly two-dimensional exchange experiments. The resonances of the nicotinamide protons of the coenzyme and the pteridine 7-proton of the folate have different chemical shifts in the three conformations, in some cases differing by more than 1 ppm. Comparison of the COSY spectra of the complex at low pH (conformation I) and high pH (conformations IIa and IIb) with that of the enzyme-methotrexate-NADP+ complex shows only slight differences in the conformation of the protein. The pattern of chemical shift changes in the ligand and the protein indicates that the structural differences are localized within the active site of the enzyme. Nucear Overhauser effects (NOEs) are observed between the nicotinamide 5- and 6-protons and the methyl resonance of Thr 45 at both low and high pH, indicating that there is no major movement of the nicotinamide ring. By contrast, NOEs are observed between the pteridine 7-proton and the methyl protons of Leu 19 and Leu 27 in conrmations I and IIa but not in conformation IIb. A model is proposed that can qualitatively account for the observed NOEs and ligand 1H chemical shifts. In this model, the folate pteridine ring in conformations I and IIa is bound in a way similar to that observed for the pteridine ring of methotrexate in the crystal structure of the enzyme-methotrexate-NADPH complex. In conformation IIb, however, it has a quite different orientation in the binding site, related to that in states I and IIa by a rotation of about 180.degree. about an axis approxiametly along the C2.sbd.NH2 bond. This latter orientation would account for the observed stereochemistry of reduction of folate by the enzyme. It thus appears that folate is able to bind to the enzyme both in a productive and in a quite different nonproductive mode, while the inhibitor methotrexate binds only in the nonproductive mode.