MULTINUCLEAR NMR CHARACTERIZATION OF 2 COEXISTING CONFORMATIONAL STATES OF THE LACTOBACILLUS-CASEI DIHYDROFOLATE-REDUCTASE TRIMETHOPRIM NADP+ COMPLEX
MULTINUCLEAR NMR CHARACTERIZATION OF 2 COEXISTING CONFORMATIONAL STATES OF THE LACTOBACILLUS-CASEI DIHYDROFOLATE-REDUCTASE TRIMETHOPRIM NADP+ COMPLEX
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DOI：
10.1021/bi00315a032
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发表时间：
1984-01-01
期刊：
影响因子：
2.9
通讯作者：
CLORE, GM
中科院分区：
文献类型：
--
作者：
BIRDSALL, B;BEVAN, AW;CLORE, GM
干酪乳杆菌二氢叶酸还原酶与甲氧苄啶和NADP+的复合物在溶液中以大约等量的2种缓慢相互转化的构象状态的混合物存在。通过多核核磁共振实验，提出了部分结构模型。在500 MHz下的1H NMR谱表明，7个组氨酸残基中的6个在2种构象之间的环境不同。的特征1H和31 P的化学位移的辅酶的核在2构象的复合物是相同的类似的复合物形成的甲氧苄啶类似物的数量，表明2构象的性质是相同的，在每种情况下。焦磷酸盐31 P共振已被分配到2个构象，和31 P光谱的整合表明，在与各种甲氧苄啶类似物的复合物中，构象I与构象II的比率从0.4到2.3不等，甲氧苄啶复合物本身的比率为1.2。转移NOE [核Overhauser效应]实验，以及1H和13 C化学位移，表明在复合物的构象II中，辅酶的烟酰胺环已经从酶表面摆动到溶液中。这是通过改变焦磷酸部分的构象而实现的。在构象I中，烟酰胺环保持与酶结合。13 C和15 N实验表明，甲氧苄啶质子化的N1在这两种构象的三元复合物。根据环电流效应对甲氧苄啶的1H位移的分析表明，在三元复合物的构象I中，甲氧苄啶保留了与其二元复合物中相同的构象，但13 C、15 N和19 F [使用2，4-二氨基-5-甲基-1，2，4-三唑-1-基]保留了与其二元复合物中相同的构象。（三、5-二甲氧基-4-氟苄基）嘧啶]实验表明，嘧啶环和苄基环的环境都受到辅酶邻近性的影响。关于复合物的构象II中的抑制剂的构象的信息较少，但其环境与二元酶-抑制剂复合物中的相似。这2个构象的酶的存在的影响，了解NADP+和甲氧苄啶之间的结合的协同性进行了简要的讨论。
The complex of Lactobacillus casei dihydrofolate reductase with trimethoprim and NADP+ exists in solution as a mixture of approximately equal amounts of 2 slowly interconverting conformational states. By multinuclear NMR experiments, a partial structural model has been proposed. 1H NMR spectra at 500 MHz show that the environments of 6 of the 7 histidine residues differ between the 2 conformations. The characteristic 1H and 31P chemical shifts of nuclei of the coenzyme in the 2 conformations of the complex are identical in analogous complexes formed with a number of trimethoprim analogues, indicating that the nature of the 2 conformations is the same in each case. The pyrophosphate 31P resonances have been assigned to the 2 conformations, and integration of the 31P spectrum shows that the ratio of conformation I to conformation II varies from 0.4 to 2.3 in the complexes with the various trimethoprim analogues, the ratio for the trimethoprim complex itself being 1.2. Transferred NOE [nuclear Overhauser effect] experiments, together with the 1H and 13C chemical shifts, indicate that in conformation II of the complex the nicotinamide ring of the coenzyme has swung away from the enzyme surface into solution. This is made possible by changes in the conformation of the pyrophosphate moiety. In conformation I, the nicotinamide ring remains bound to the enzyme. 13C and 15N experiments show that trimethoprim is protonated on N1 in both conformations of the ternary complex. Analysis of the 1H shifts of trimethoprim in terms of ring current effects shows that in conformation I of the ternary complex trimethoprim retains the same conformation as in its binary complex, but 13C, 15N, and 19F [using 2,4-diamino-5-(3,5-dimethoxy-4-fluorobenzyl)pyrimidine] experiments show that the environment of both the pyrimidine ring and benzyl ring is affected by the proximity of the coenzyme. Less information is available about the conformation of the inhibitor in conformation II of the complex, but its environment is similar to that in the binary enzyme-inhibitor complex. The implications of the existence of these 2 conformations of the enzyme for understanding cooperativity in binding between NADP+ and trimethoprim are briefly discussed.