Methenyltetrahydrofolate cyclohydrolase is rate limiting for the enzymatic conversion of 10-formyltetrahydrofolate to 5,10-methylenetetrahydrofolate in bifunctional dehydrogenase-cyclohydrolase enzymes
Methenyltetrahydrofolate cyclohydrolase is rate limiting for the enzymatic conversion of 10-formyltetrahydrofolate to 5,10-methylenetetrahydrofolate in bifunctional dehydrogenase-cyclohydrolase enzymes
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DOI：
10.1021/bi971906t
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发表时间：
1998-01-27
期刊：
影响因子：
2.9
通讯作者：
MacKenzie, RE
中科院分区：
文献类型：
--
作者：
Pawelek, PD;MacKenzie, RE
三个亚甲基四氢叶酸脱氢酶环化水解酶（D/C）酶（人细胞质三功能酶的NADP依赖性双功能结构域，人线粒体NAD依赖性双功能酶，和NAD（P）依赖性双功能酶发光菌）的动力学性质进行了测定，在正向和反向。在正向方向上，这些酶具有广泛不同的k（cat）C/k（cat）D比率，但所有通道由D活性与C活性产生的亚甲基H（4）叶酸具有大致相同的效率。在正向和反向脱氢酶测定中均观察到人NADP依赖性酶的氘同位素效应，这与氢化物转移是次甲基和亚甲基H（4）叶酸相互转化的速率限制一致。然而，没有观察到整体逆反应（甲酰H（4）叶酸到亚甲基H（4）叶酸）的动力学同位素效应。我们设计了一种测定法来测量不依赖于脱氢酶的反向环化水解酶活性，并确定每种酶的k（cat）（总反向）约等于其反向环化水解酶活性的k（cat）。因此，在整个逆反应中的限速步骤不是脱氢酶的氢化物转移，而是环化水解酶催化的亚甲基H（4）叶酸的产生。NADP依赖性D/C和P. phosphoreum酶的反向环化水解酶活性，但线粒体NAD依赖性酶的活性，可以通过添加2 '，5'-ADP刺激2倍。结果表明，人NADP依赖性和P. phosphoreum酶的环水解酶被优化以在结合的辅酶存在下催化逆反应。这些结果意味着在逆反应中由环化水解酶产生的亚甲基H（4）叶酸基本上全部被引导至脱氢酶。
The kinetic properties of three methylenetetrahydrofolate dehydrogenase-cyclohydrolase (D/C) enzymes (the NADP-dependent bifunctional domain of the human cytoplasmic trifunctional enzyme, the human mitochondrial NAD-dependent bifunctional enzyme, and the NAD(P)-dependent bifunctional enzyme from Photobacterium phosphoreum) were determined in both forward and reverse directions. Ln the forward direction, the enzymes possess widely different ratios of k(cat) C/k(cat) D, but all channel methenylH(4)folate produced by the D activity to the C activity with approximately the same efficiency. A deuterium isotope effect is observed with the human NADP-dependent enzyme in both forward and reverse dehydrogenase assays, consistent with hydride transfer being rate limiting for the interconversion of methenyl- and methyleneH(4)folate. However, no kinetic isotope effect is observed for the overall reverse reaction (formylH(4)folate to methyleneH(4)folate). We devised an assay to measure the reverse cyclohydrolase activity independent of the dehydrogenase, and determined that the k(cat) (overall reverse) for each enzyme is approximately equal to the k(cat) for its reverse cyclohydrolase activity. Therefore, the rate-limiting step in the overall reverse reaction is not hydride transfer by the dehydrogenase, but the production of methenylH(4)folate catalyzed by the cyclohydrolase. The reverse cyclohydrolase activities of the NADP-dependent D/C and the P. phosphoreum enzymes, but not the mitochondrial NAD-dependent enzyme, can be stimulated 2-fold by the addition of 2',5'-ADP. The results suggest that the cyclohydrolases of the human NADP dependent and P. phosphoreum enzymes are optimized to catalyze the reverse reaction in the presence of bound coenzyme. These results imply that essentially all of the methenylH(4)folate produced by the cyclohydrolase in the reverse reaction is channeled to the dehydrogenase.