Crowders Steal Dihydrofolate Reductase Ligands through Quinary Interactions.
Crowders Steal Dihydrofolate Reductase Ligands through Quinary Interactions.
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Crowders 通过五元相互作用窃取二氢叶酸还原酶配体。
DOI：
10.1021/acs.biochem.8b01110
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发表时间：
2019
期刊：
影响因子：
2.9
通讯作者：
Howell,ElizabethE
中科院分区：
文献类型：
--
作者：
DuffJr,MichaelR;Desai,Nidhi;Craig,MichaelA;Agarwal,PratulK;Howell,ElizabethE
二氢叶酸还原酶 (DHFR) 使用 NADPH 作为辅助因子将二氢叶酸 (DHF) 还原为四氢叶酸。由于其在一碳代谢中的作用，染色体 DHFR 是抗菌药物甲氧苄啶的靶标。对甲氧苄啶的耐药性导致产生 II 型 DHFR，其在结构上与染色体酶靶标不相关。由于其代谢意义，了解 DHFR 动力学和在更像细胞的条件下的配体结合行为（其中总大分子浓度可能高达 300 mg/mL）非常重要。在大分子共溶质存在的情况下，研究了药物靶标（染色体大肠杆菌DHFR）和耐药酶（R67 DHFR）的进展曲线动力学和配体结合特性。对 NADPH 氧化和与两种 DHFR 的结合有不同的影响，一些共溶质增加亲和力，另一些则减弱结合。然而，在所有共溶质存在的情况下，DHF 结合和两种 DHFR 的减少都会减少。配体结合的减少主要归因于与大分子的弱结合，而不是对 DHFR 的拥挤效应。计算机模拟发现两种配体与几种蛋白质之间存在微弱、短暂的相互作用。蛋白质共溶质的净电荷与对 NADP+ 结合的影响相关，接近中性和带正电荷的蛋白质对结合具有更不利的影响。对于 DHF 结合，效果与蛋白质拥挤器上结合袋的大小更相关。 DHFR 配体和蛋白质之间的这些非特异性相互作用预示着 DHFR 的体内效率可能远低于其体外效率的预期。
Dihydrofolate reductase (DHFR) reduces dihydrofolate (DHF) to tetrahydrofolate using NADPH as a cofactor. Due to its role in one carbon metabolism, chromosomal DHFR is the target of the antibacterial drug, trimethoprim. Resistance to trimethoprim has resulted in a type II DHFR that is not structurally related to the chromosomal enzyme target. Because of its metabolic significance, understanding DHFR kinetics and ligand binding behavior in more cell-like conditions, where the total macromolecule concentration can be as great as 300 mg/mL, is important. The progress-curve kinetics and ligand binding properties of the drug target (chromosomalE. coliDHFR) and the drug resistant (R67 DHFR) enzymes were studied in the presence of macromolecular cosolutes. There were varied effects on NADPH oxidation and binding to the two DHFRs, with some cosolutes increasing affinity and others weakening binding. However, DHF binding and reduction in both DHFRs decreased in the presence of all cosolutes. The decreased binding of ligands is mostly attributed to weak associations with the macromolecules, as opposed to crowder effects on the DHFRs. Computer simulations found weak, transient interactions for both ligands with several proteins. The net charge of protein cosolutes correlated with effects on NADP+binding, with near neutral and positively charged proteins having more detrimental effects on binding. For DHF binding, effects correlated more with the size of binding pockets on the protein crowders. These nonspecific interactions between DHFR ligands and proteins predict that thein vivoefficiency of DHFRs may be much lower than expected from theirin vitrorates.