Isolation and characterization of a novel eukaryotic monofunctional NAD(+)-dependent 5,10-methylenetetrahydrofolate dehydrogenase.
Isolation and characterization of a novel eukaryotic monofunctional NAD(+)-dependent 5,10-methylenetetrahydrofolate dehydrogenase.
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新型真核单功能 NAD() 依赖性 5,10-亚甲基四氢叶酸脱氢酶的分离和表征。
DOI：
10.1021/bi00482a020
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发表时间：
1990
期刊：
影响因子：
2.9
通讯作者：
Appling,DR
中科院分区：
文献类型：
--
作者：
Barlowe,CK;Appling,DR
德克萨斯大学化学系和克莱顿基金会生化研究所，奥斯汀，德克萨斯州 78712 收稿日期：1990 年 2 月 27 日；修订稿于 1990 年 4 月 16 日收到 摘要：一种 NAD+ 依赖性 5, 10-亚甲基四氢叶酸 (THF) 脱氢酶已从酿酒酵母中纯化至均质。纯化的酶表现出5.4单位mg-1的最终比活性，并由十二烷基硫酸钠凝胶电泳测定的表观M i = 33 000-38 000的单一蛋白质表示。通过凝胶过滤测定天然Mr= 64 000，表明同二聚体亚基结构。与辛二酰亚胺酸二甲酯的交联实验证实了二聚体结构。该酶对 NAD+ 具有特异性，并且不依赖于 Mg2+ 来发挥活性。正向反应初速度动力学与顺序反应机制一致。对于该模型，NAD+ 和 (6/?, 5)-5, 10-亚甲基-THF 的 Km 值分别为 1.6 和 0.06 mM。与之前描述的所有其他真核5, 10-亚甲基-THF脱氢酶相比，纯化的酶显然是单功能的，具有不可检测的5, 10-亚甲基-THF环水解酶和10-甲酰基-THF合成酶活性。酵母的亚细胞分级表明该酶是细胞质的，在线粒体中未检测到 NAD+ 依赖性 5, 10-亚甲基-THF 脱氢酶。在所有检查的酵母菌株中，从滞后期到稳定期的所有生长阶段都发现了这种活性。叶酸介导的一碳代谢在几个主要细胞过程中发挥着重要作用，包括核酸生物合成、线粒体和叶绿体蛋白质生物合成、氨基酸生物合成和转化以及维生素代谢。利用这些单碳单元的途径的多样性取决于生物体改变附着在单碳单元上的碳单元的氧化态的能力。
Department of Chemistry and Clayton Foundation Biochemical Institute, The University of Texas, Austin, Texas 78712 Received February 27, 1990; Revised Manuscript Received April 16, 1990 abstract: An NAD+-dependent 5, 10-methylenetetrahydrofolate (THF) dehydrogenase has been purified to homogeneity from the yeast Saccharomyces cerevisiae. The purified enzyme exhibits a final specific activity of 5.4 units mg-1 and is represented by a single protein of apparent M,= 33 000-38 000 as determined by sodium dodecyl sulfate gel electrophoresis. A native Mr= 64 000 was determined by gel filtration, suggesting a homodimer subunit structure. Cross-linking experiments with dimethyl suberimidate confirmed the dimeric structure. The enzyme is specific for NAD+ and is not dependent on Mg2+ for activity. The forward reaction initial velocity kinetics are consistent with a sequential reaction mechanism. With this model, Km values for NAD+ and (6/?, 5)-5, 10-methylene-THF are 1.6 and 0.06 mM, respectively. In contrast to all other previously described eukaryotic 5, 10-methylene-THF dehydrogenases, the purified enzyme is apparently monofunctional, with undetectable 5, 10-methenyl-THF cyclohydrolase and 10-formyl-THF synthetase activities. Subcellularfractionation of yeast indicates the enzyme is cytoplasmic, with no NAD+-dependent 5, 10-methylene-THF dehydrogenase detectable in mitochondria. The activity was found in all yeast strains examined, at all stages of growth from the lag phase through the stationary phase.Folate-mediated one-carbon metabolism plays an essential role in several majorcellular processes including nucleic acid biosynthesis, mitochondrial and chloroplast protein biosyn-thesis, amino acid biosynthesis and conversions, and vitamin metabolism. The variety of pathways thatutilize these one-carbon units is dependent upon the ability of the organism to vary the oxidation state of the carbon unit attached to the