Tales of Dihydrofolate Binding to R67 Dihydrofolate Reductase.
Tales of Dihydrofolate Binding to R67 Dihydrofolate Reductase.
复制标题
DOI：
10.1021/acs.biochem.5b00981
复制
发表时间：
2016-01-12
期刊：
影响因子：
2.9
通讯作者：
Howell, Elizabeth E.
中科院分区：
文献类型：
--
作者：
Duff, Michael R., Jr.;Chopra, Shaileja;Strader, Michael Brad;Agarwal, Pratul K.;Howell, Elizabeth E.
同四聚体R67二氢叶酸还原酶具有222个对称性和单一的活性中心孔。这种情况导致杂乱的结合部位，该结合部位要么容纳底物二氢叶酸(DHF)，要么容纳辅因子NADPH。NADPH与蛋白质的相互作用更直接，因为它比底物大。相比之下，通过核磁共振和结晶学的监测，DHF的对氨基苯甲酰谷氨酸尾在结合时是无序的。为了探索较小的活性部位体积(这应该会降低限制效应导致的尾部无序程度)是否会改变稳态速率，构建了使半孔体积减少∼35%的不对称突变。仅观察到对kcat的轻微影响。为了继续探索尾部无序在催化中的作用，我们进行了R67dhfr和叶酸之间的1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide-mediated交联。两个叶酸，一个四聚体的复合体导致酶活性的丧失，其中蛋白质中的两个对称相关的K32残基与两个结合的叶酸的羧酸交联。被束缚的叶酸可以减少，尽管≤速率降低了30倍，这表明动力学降低和/或用于催化的交联叶酸的位置不佳。抑制K32附近二氢叶酸尾巴的计算机模拟表明，交联仍然允许对氨基苯甲酰环的移动，从而允许反应发生。最后，合成了双乙二胺-α，γ-酰胺叶酸加合物；谷氨酸尾部的两个带负电荷的羧基都被带正电的胺取代。该加合物的KI是叶酸的∼的9倍。这些不同的结果表明，叶酸尾部紊乱之间存在平衡，叶酸尾部紊乱有助于酶结合底物，而动力学有助于催化。
Homotetrameric R67 dihydrofolate reductase possesses 222 symmetry and a single active site pore. This situation results in a promiscuous binding site that accommodates either the substrate, dihydrofolate (DHF), or the cofactor, NADPH. NADPH interacts more directly with the protein as it is larger than the substrate. In contrast, the p-aminobenzoyl-glutamate tail of DHF, as monitored by nuclear magnetic resonance and crystallography, is disordered when bound. To explore whether smaller active site volumes (which should decrease the level of tail disorder by confinement effects) alter steady state rates, asymmetric mutations that decreased the half-pore volume by ∼35% were constructed. Only minor effects on kcat were observed. To continue exploring the role of tail disorder in catalysis, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide-mediated cross-linking between R67 DHFR and folate was performed. A two-folate, one-tetramer complex results in the loss of enzyme activity where two symmetry-related K32 residues in the protein are cross-linked to the carboxylates of two bound folates. The tethered folate could be reduced, although with a ≤30-fold decreased rate, suggesting decreased dynamics and/or suboptimal positioning of the cross-linked folate for catalysis. Computer simulations that restrain the dihydrofolate tail near K32 indicate that cross-linking still allows movement of the p-aminobenzoyl ring, which allows the reaction to occur. Finally, a bis-ethylene-diamine-α,γ-amide folate adduct was synthesized; both negatively charged carboxylates in the glutamate tail were replaced with positively charged amines. The Ki for this adduct was ∼9-fold higher than for folate. These various results indicate a balance between folate tail disorder, which helps the enzyme bind substrate while dynamics facilitates catalysis.
登录
查看更多内容
影响因子：
2.9
作者：
BACCANARI, D;PHILLIPS, A;BURCHALL, J
通讯作者：
BURCHALL, J
影响因子：
2.9
作者：
Krahn, Joseph M.;Jackson, Michael R.;London, Robert E.
通讯作者：
London, Robert E.
影响因子：
2.9
作者：
Feng, Jian;Grubbs, Jordan;Howell, Elizabeth E.
通讯作者：
Howell, Elizabeth E.
影响因子：
4.8
作者：
Hicks, SN;Smiley, RD;Howell, EE
通讯作者：
Howell, EE
影响因子：
2.9
作者：
GILLES, MA;HUDSON, AQ;BORDERS, CL
通讯作者：
BORDERS, CL