Mechanistic studies of a flavin-dependent thymidylate synthase
Mechanistic studies of a flavin-dependent thymidylate synthase
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DOI：
10.1021/bi0490439
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发表时间：
2004-08-17
期刊：
影响因子：
2.9
通讯作者：
Kohen, A
中科院分区：
文献类型：
--
作者：
Agrawal, N;Lesley, SA;Kohen, A
编码胸苷酸合成酶（TS）的ThyA基因在大量细菌的基因组中不存在，包括几种人类病原体。这些细菌中的许多也缺乏二氢叶酸还原酶（DHFR）和胸苷激酶的基因，并且完全依赖于胸苷酸合成的替代酶。Thy 1编码黄素依赖性TS（FDTS，以前表示为TSCP），并且与经典TS基因没有序列同源性。本文介绍了海栖热袍菌（TM 0449）FDTS的机理研究。几个同位素标记实验揭示了催化反应的细节，并提出了与实验数据一致的化学机制。该反应通过乒乓机制进行，其中烟酰胺结合和释放先于氧化半反应。该酶主要对烟酰胺（NADPH）具有pro-R特异性，其氧化是整个催化级联反应的限速步骤。酶结合的黄素减少与同位素效应25（与H-隧道）和交换质子与溶剂之前，减少中间体亚甲基。建立了定量分析方法，并测定了动力学参数。一个显着的NADPH底物抑制和大Km合理的缓慢活动，这种酶在过去的报告。这些和其他调查结果进行比较，希望经典TS（ThyA）催化的动力学和分子机制。FDTS提出的机制与经典TS的机制之间的差异是惊人的，并引发了这样的概念，即基于机制的药物将选择性地抑制FDTS，并且对人类（和其他真核生物）TS没有太大影响。由于TS活性对DNA复制至关重要，FDTS的独特机制使其成为抗生素药物开发的有吸引力的靶标。
The ThyA gene that encodes for thymidylate synthase (TS) is absent in the genomes of a large number of bacteria, including several human pathogens. Many of these bacteria also lack the genes for dihydrofolate reductase (DHFR) and thymidine kinase and are totally dependent on an alternative enzyme for thymidylate synthesis. Thy1 encodes flavin-dependent TS (FDTS, previously denoted as TSCP) and shares no sequence homology with classical TS genes. Mechanistic studies of a FDTS from Thermotoga maritima (TM0449) are presented here. Several isotopic labeling experiments reveal details of the catalyzed reaction, and a chemical mechanism that is consistent with the experimental data is proposed. The reaction proceeds via a ping-pong mechanism where nicotinamide binding and release precedes the oxidative half-reaction. The enzyme is primarily pro-R specific with regard to the nicotinamide (NADPH), the oxidation of which is the rate-limiting step of the whole catalytic cascade. An enzyme-bound flavin is reduced with an isotope effect of 25 (consistent with H-tunneling) and exchanges protons with the solvent prior to the reduction of an intermediate methylene. A quantitative assay was developed, and the kinetic parameters were measured. A significant NADPH substrate inhibition and large Km rationalized the slow activity reported for this enzyme in the past. These and other findings are compared wish classical TS (ThyA) catalysis in terms of kinetic and molecular mechanisms. The differences between the FDTS proposed mechanism and that of the classical TS are striking and invoke the notion that mechanism-based drugs will selectively inhibit FDTS and will not have much effect on human (and other eukaryotes) TS. Since TS activity is essential to DNA replication, the unique mechanism of FDTS makes it an attractive target for antibiotic drug development.