Enzymatic characterization of human mitochondrial C1-tetrahydrofolate synthase
Enzymatic characterization of human mitochondrial C1-tetrahydrofolate synthase
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DOI：
10.1016/j.abb.2005.08.007
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发表时间：
2005-10-15
影响因子：
3.9
通讯作者：
Appling, DR
中科院分区：
文献类型：
--
作者：
Walkup, AS;Appling, DR
人线粒体C-1-四氢叶酸（THF）合酶的同工酶先前被鉴定为与人细胞质C-1-THF合酶相似。迄今为止，从酵母到人的所有C-1-THF脱氢酶都是三功能的，含有5，10-亚甲基-THF脱氢酶、5，10-亚甲基-THF环化水解酶和10-甲酰基-THF合成酶的活性。在这里，我们报告的重组人线粒体同工酶的酶特性。纯化的人线粒体C-1-THF合成酶蛋白的酶测定显示仅存在10-甲酰基-THF合成酶活性。凝胶过滤和交联研究表明，人线粒体C-1-THF合酶在溶液中以同源二聚体形式存在。使用（6 R，S）-H-4-PteGlu（1）、（6 R，S）-H-4-PteGlu（3）和（6 R，S）-H-4-PteGlu（5）底物进行10-甲酰基-THF合成酶活性的稳态动力学表征。（6 R，S）-H-4-PteGlu（n）K-m从单谷氨酸盐的大于500 μ m降至15 μ m。和3.6 μ m。当使用THF聚谷氨酸盐时，甲酸盐和ATP的Km值也降低。当使用（6 R，S）-H-4-PteGlu（5）代替（6 R，S）-H-4-PteGlu（1）（c）2005 Elsevier Inc. All rights reserved.
A human mitochondrial isozyme of C-1-tetrahydrofolate (THF) synthase was previously identified by its similarity to the human cytoplasmic C-1-THF synthase. All C-1-THF synthases characterized to date, from yeast to human, are trifunctional, containing the activities of 5,10-methylene-THF dehydrogenase, 5,10-methenyl-THF cyclohydrolase, and 10-formyl-THF synthetase. Here we report on the enzymatic characterization of the recombinant human mitochondrial isozyme. Enzyme assays of purified human mitochondrial C-1-THF synthase protein revealed only the presence of 10-formyl-THF synthetase activity. Gel filtration and crosslinking studies indicated that human mitochondrial C-1-THF synthase exists as a homodimer in solution. Steady-state kinetic characterization of the 10-formyl-THF synthetase activity was performed using (6R,S)-H-4-PteGlu(1), (6R,S)-H-4-PteGlu(3), and (6R,S)-H-4-PteGlu(5) substrates. The (6R,S)-H-4-PteGlu(n) K-m dropped from greater than 500 mu m for the monoglutamate to 15 mu m. and 3.6 mu m for the tri- and pentaglutamates, respectively. The K-m values for formate and ATP also are lowered when THF polyglutamates are used. The formate K-m, dropped 79-fold and the ATP K-m dropped more than 5-fold when (6R,S)-H-4-PteGlu(5) was used as the substrate in place of (6R,S)-H-4-PteGlu(1) (c) 2005 Elsevier Inc. All rights reserved.