The Role of Arginine 28 in Catalysis by Dihydrofolate Reductase from the Hyperthermophile Thermotoga maritima
The Role of Arginine 28 in Catalysis by Dihydrofolate Reductase from the Hyperthermophile Thermotoga maritima
复制标题
DOI：
10.1002/cbic.200900465
复制
发表时间：
2009-11-02
期刊：
影响因子：
3.2
通讯作者：
Allemann, Rudolf K.
中科院分区：
文献类型：
--
作者：
Loveridge, E. Joel;Maglia, Giovanni;Allemann, Rudolf K.
二氢叶酸还原酶（DHFR）以还原性烟酰胺腺嘌呤二核苷酸磷酸（NADPH）为辅助因子，催化7,8 -二氢叶酸（H2F）还原为5,6,7,8 -四氢叶酸（H4F）。在反应中，氢化物从NADPH转移到底物的蝶呤环的C6上，底物的N5被质子化。H4F在各种氧化状态下作为单碳单位的载体，是胸苷酸、嘌呤、几种氨基酸以及原核生物泛酸的合成所必需的来自超嗜热菌Thermotoga martima （TmDHFR）的DHFR是唯一以二聚体形式存在的染色体DHFR，尽管其三级结构与来自大肠杆菌（EcDHFR）的单体DHFR非常相似TmDHFR催化反应的速率远低于EcDHFR[4,5]，这被认为是由于二聚化导致TmDHFR失去灵活性的结果。[2,4] [4] TmDHFR的熔化温度比EcDHFR高约308℃，是迄今为止分离出的最耐热的DHFR。[5-7]我们最近表明，TmDHFR的二聚体结构对其高热稳定性至关重要。但对其催化活性影响不显著；TmDHFR的一个单体单残基突变体具有与野生型酶相似的催化性能因此，需要对TmDHFR较低速率的其他解释，包括在活性位点的底物结合区存在精氨酸28（图1），[4]TmDHFR的活性位点更多地暴露于溶剂，[2]以及TmDHFR和EcDHFR遵循细微不同的底物还原机制的观察。许多精氨酸残基通常存在于活性位点周围，以结合底物的谷氨酸“尾巴”（图1），但在细菌DHFRs中，在底物N5附近存在Arg28是不寻常的。这个位置通常由亮氨酸或苯丙氨酸残基占据。Arg28降低tmdhfr催化反应速率的最可能机制是由于其正电荷而不利于底物的质子化。TmDHFR中的氢化物转移和稳态转换均表现出s型的pH依赖性，表观pKa约为6.0 [4]的
Dihydrofolate reductase (DHFR) catalyses the reduction of 7, 8-dihydrofolate (H2F) to 5, 6, 7, 8-tetrahydrofolate (H4F) by using reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor. In the reaction, hydride is transferred from NADPH to C6 of the pterin ring of the substrate and N5 of the substrate is protonated. H4F acts as a carrier of one-carbon units in various oxidation states and is required for the synthesis of thymidylate, purines, several amino acids and, in prokaryotes, pantothenate.[1] DHFR from the hyperthermophile Thermotoga maritima (TmDHFR) is the only chromosomal DHFR that exists as a dimer,[2] although its tertiary structure is very similar to that of the monomeric DHFRs such as that from E. coli (EcDHFR).[3] The rates of the TmDHFR-catalysed reaction are considerably lower than those of EcDHFR [4, 5] and this had been proposed to be a consequence of the loss of flexibility in TmDHFR as a result of dimerisation.[2, 4] With a melting temperature approximately 308C above that of EcDHFR,[4] TmDHFR is the most thermostable DHFR isolated to date.[5–7] We have recently shown that the dimeric structure of TmDHFR is critical for its high thermostability. However, it does not significantly affect its catalytic activity; a monomeric single residue mutant of TmDHFR had catalytic properties that were similar to those of the wild-type enzyme.[8] Alternative explanations for the lower rates of TmDHFR are hence required and include the presence of arginine 28 in the substrate-binding region of the active site (Figure1),[4] the more solventexposed active site of TmDHFR,[2] and the observation that TmDHFR and EcDHFR follow subtly different mechanisms for reduction of the substrate.[9]A number of arginine residues are generally present around the active site to bind the glutamate “tail” of the substrate (Figure 1), but the presence of Arg28 in close proximity to N5 of the substrate is unusual in bacterial DHFRs. This position is normally occupied by a leucine or phenylalanine residue. The most likely mechanism by which Arg28 could reduce the rate of the TmDHFR-catalysed reaction is by disfavouring protonation of the substrate due to its positive charge. Both hydride transfer and steady-state turnover in TmDHFR show sigmoidal pH dependences with an apparent pKa of around 6.0.[4] The