二氢叶酸还原酶可将二氢叶酸转化为四氢叶酸,四氢叶酸参与从头合成嘌呤、胸苷酸和某些氨基酸的甲基转移反应。 虽然研究发现功能性的二氢叶酸还原酶基因定位于5号染色体,但在不同的染色体上已鉴定出多个无内含子加工的假基因或二氢叶酸还原酶类似的基因。
由二氢叶酸还原酶催化的反应。
四氢叶酸合成路线。
研究发现,在所有生物体中,DHFR在调节细胞中四氢叶酸的含量方面起着关键作用。四氢叶酸及其衍生物对嘌呤和胸苷酸的合成反应必不可少,而嘌呤和胸苷酸的合成对细胞增殖和细胞生长至关重要。
DHFR在核酸前体的合成中发挥着核心作用,且研究表明完全缺乏DHFR的突变细胞需要氨基酸中的甘氨酸和胸苷才能生长。[8] DHFR也被证明是一种参与将二氢生物蝶呤中还原为四氢生物蝶呤的补救反应的酶。[9]
二氢叶酸还原酶催化二氢叶酸还原为四氢叶酸。
二氢叶酸还原酶(DHFR)催化氢原子从NADPH转移到二氢叶酸,伴随质子化反应生成四氢叶酸。[10] 最后,二氢叶酸被还原成四氢叶酸,NADP被氧化成NADP+。 Met20和活性位点附近其他环的高灵敏性在促进产物四氢叶酸的释放中起作用。特别是Met20环有助于稳定NADPH的烟酰胺环,促进氢化物从NADPH转移到二氢叶酸。[10]
这种酶的机理是逐步的和稳态随机的。具体而言,催化反应始于NADPH和附着于酶结合位点的底物,接着是质子化反应和氢化物从辅酶NADPH转移到底物。然而,后两个步骤不会在同一过渡状态下同时发生。[10][11] 在一项结合计算和实验方法的研究中,刘等人提出质子化步骤先于氢化物转移的结论。[12]
封闭的结构用红色表示,在催化方案中用绿色描绘了封闭结构。 在该结构中,DHF和THF显示为红色,NADPH显示为黄色,Met20残基显示为蓝色。
二氢叶酸还原酶(DHFR)的酶促机理被证明是依赖于酸碱度的,特别是氢化物转移步骤,因为酸碱度的变化被证明对活性位点的静电作用和其残基的电离状态有显著的影响。[12] 底物目标位置上氮的酸性对底物与酶结合位点的结合过程很重要,即使该结合位点与水直接接触,它也被证明是疏水的。[10][13] Asp27是结合位点上唯一带电的亲水性残基,Asp27上电荷的中和可能改变酶的pKa。Asp27辅助底物质子化并且能够将底物限制在有利于氢化物转移的构象中,在催化机制中起着关键作用。[14][10][13] 质子化步骤被证明与烯醇互变异构有关,尽管这种转化并不利于质子转移。[11] 水分子被证明参与质子化步骤。[15][16][17] 水分子进入酶的活性位点是由Met20环促进的。[18]
二氢叶酸还原酶(DHFR)催化循环过程包括五个重要的中间体:全酶(E:NADPH)、米氏复合物(E:NADPH:DHF)、三元产物复合物(E:NADP+:THF)、四氢叶酸二元复合物(E:THF)和THF NADPH复合物(E:NADPH:THF)。产物(THF)从E:NADPH:THF到E:NADPH的解离步骤是稳态转换的决速步。[14]
构象变化在二氢叶酸还原酶(DHFR)的催化机理中至关重要。[19] DHFR的Met20环能够打开、关闭或封闭活动部位。[16][10] 相应地,分类为打开、关闭和阻塞的三种不同构象都归属于Met20。此外,由于其表征结果不够清晰,Met20也还存在其他扭曲构象。[16] 在连接三种产物的中间体中观察到Met20环的封闭构象,其中酰胺环从活性位点处封闭。这种构象特征解释了NADP+被NADPH取代先于产物解离的事实。因此,下一轮反应可以在底物结合时发生。[14]
EcDHFR和R67 DHFR的反应动力学比较
大肠杆菌和R67 DHFR中底物结合的结构差异
R67 二氢叶酸还原酶因其独特的结构和催化特性而被广泛研究。R67 二氢叶酸还原酶(R67 DHFR)是一种II型R质粒编码的二氢叶酸还原酶(DHFR),与大肠杆菌染色体二氢叶酸还原酶(DHFR)无遗传和结构关系。它是一种同源四聚体,具有222对称性,拥有一个暴露于溶剂的活性位点孔。 活性位点的这种对称性导致酶具有不同结合模式:它可以与两个具有正协同性的二氢叶酸(DHF)分子或两个具有负协同性的NADPH分子结合,或者一个底物加其中一个分子,但只有后者具有催化活性。[20] 与大肠杆菌染色体二氢叶酸还原酶(DHFR)相比,它在结合二氢叶酸(DHF)和NADPH方面具有更高的Km值。更低级的催化动力学表明氢化物转移才是速率决定步骤,而不是产物(THF)释放。[21]在二氢叶酸还原酶(R67 DHFR)结构中,同源四聚体形成活性位点孔。在催化过程中,二氢叶酸(DHF)和NADPH从相反的位置进入孔隙。NADPH烟酰胺环和DHF蝶啶环之间的π-π堆积相互作用将活性位点上的两种反应物紧密相连。然而,结合状态下观察到二氢叶酸(DHF)分子的对氨基苯甲酰谷氨酸尾部的柔韧性可以促进过渡态的形成。[22]
由于快速分裂的细胞需要叶酸来合成胸腺嘧啶,因此可以利用该效果用于治疗。
DHFR既可作为癌症治疗的靶标,也是抵抗细菌感染的潜在靶标。 DHFR负责调控细胞中四氢叶酸的水平,且DHFR的抑制可以限制细胞生长与增殖,这是癌症与细菌感染的主要特征。甲氨蝶呤是DHFR的竞争性抑制剂,是具有抑制DHFR功能的抗癌药物之一。[26] 其他药物包括甲氧苄氨嘧啶和乙胺嘧啶。这三种药物被广泛应用于抗肿瘤与抗菌。[27] 通常靶向DHFR,尤其是细菌DHFR的其他类化合物属于例如二氨基蝶呤、二氨基三嗪、二氨基吡咯喹啉、二苯乙烯、查尔酮、脱氧苯偶姻等类型,这里略举数例。[28]
甲氧苄啶已显示出对多种革兰氏阳性细菌病原体具有活性。[29] 然而,甲氧苄啶和其他靶向DHFR药物的耐药性产生可能与多种机制有关,从而限制了其在治疗使用上的成功。[30][30][31] 抗药性可能源自DHFR基因的扩增、DHFR突变、药物摄取量减少等。无论如何,甲氧苄啶与磺胺甲𫫇唑的联合用药在近数十年来已被广泛用于抗菌。[29]
叶酸是细胞生长所必需的,[32] 叶酸代谢途径是发展癌症治疗的靶标。 DHFR就是这样一个靶标。氟尿嘧啶、阿霉素和甲氨蝶呤的治疗方案显示可以延长晚期胃癌患者的生存期。[33] 通过对DHFR抑制剂的进一步研究可以促进更多癌症治疗方法的发展。
细菌也依赖DHFR来生长与繁殖,所以对细菌DHFR有选择性的抑制剂已应用于抗菌中。[29]
用作二氢叶酸还原酶抑制剂的小分子包括二氨基喹唑啉和二氨基吡咯喹啉,[34] 二氨基嘧啶、二氨基蝶啶和二氨基三嗪等类别。[35]
经过验证,来自炭疽芽孢杆菌的二氢叶酸还原酶 (BaDHFR)是治疗传染性疾病炭疽的有效药物靶标。相对于其他物种(如大肠杆菌、金黄色葡萄球菌和肺炎链球菌)的二氢叶酸还原酶,BaDHFR对甲氧苄啶类似物的敏感性更低。 来自所有四个物种的二氢叶酸还原酶的结构序列表明,只有BaDHFR在位置96和102分别具有苯丙氨酸和酪氨酸的组合。
这两个残基(F96和Y102)导致了BaDHFR对甲氧苄啶类似物的抗药性,其也提高了动力学和催化效率。[36] 目前研究通过突变BaDHFR中的活性位点来引导新型抗叶酸抑制剂的优化。[36]
在蛋白质片段互补分析中,DHFR被用作检测蛋白质-蛋白质相互作用的工具。
缺乏DHFR的中国仓鼠卵巢细胞(CHO细胞)是生产重组蛋白最常用的细胞系。这些细胞用携带dhfr基因和重组蛋白基因的质粒在单一表达系统中转染,然后在缺乏胸苷的培养基中进行筛选。只有带有外源DHFR基因和目的基因的细胞才能存活下来。
Click on genes, proteins and metabolites below to link to respective articles.
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Fluorouracil (5-FU) Activity edit
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