Cook R J, Wagner C
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Arch Biochem Biophys. 1995 Aug 20;321(2):336-44. doi: 10.1006/abbi.1995.1403.
10-Formyltetrahydrofolate dehydrogenase (10-FTH-FDH: EC 1.5.1.6) catalyzes the NADP(+)-dependent oxidation of 10-formyltetrahydrofolate (10-HCO-H4PteGlu) to tetrahydrofolate (H4PteGlu) and CO2 and the NADP(+)-independent hydrolytic cleavage of 10-HCO-H4PteGlu to H4PteGlu and formate. 10-FTHFDH has a 485 amino acid domain at the C-terminus which is 46% identical to aldehyde dehydrogenase (ALDH: EC 1.2.1.3) and contains a conserved active site cysteine (Cys-707). 10-FTHFDH catalyzed NADP(+)-dependent oxidation of propanal and the hydrolysis of p-nitrophenyl acetate (pNPA) in a similar fashion to ALDH. Initial rate studies gave Km values of 46 and 636 microM, respectively, for NADP+ and propanal, while pNPA had a Km of 220 microM. Propanal was able to compete with 10-HCO-H4PteGlu for NADP(+)-dependent oxidation but had no effect on the NADP(+)-independent hydrolase reaction. N-Ethylmaleimide inhibited NADP(+)-dependent 10-HCO-H4PteGlu oxidation but only partially inhibited (65%) hydrolase activity. Disulfiram, a potent inhibitor of cytosolic ALDH, inhibited NADP(+)-dependent propanal oxidation by 10-FTHFDH. We propose that the dehydrogenase reaction of 10-FTHFDH has a mechanism which proceeds through thiohemiacetal and thioester intermediates, similar to that described for aldehyde dehydrogenase. 10-FTHFDH hydrolase activity was dependent on 2-mercaptoethanol and is probably an artifact of the assay system. The N-terminal domain of 10-FTHFDH shows identity to glycinamide ribonucleotide transformylase (EC 2.1.2.2) and contains a putative 10-HCO-H4PteGlu binding site but shows no GAR-TF activity. NADP(+)-dependent oxidation of 10-HCO-H4PteGlu by 10-FTHFDH was inhibited by the folate anti-metabolite, 5,10-dideazatetrahydrofolate, a known GAR-TF inhibitor.
10-甲酰四氢叶酸脱氢酶（10-FTH-FDH：EC 1.5.1.6）催化10-甲酰四氢叶酸（10-HCO-H4PteGlu）依赖NADP⁺氧化为四氢叶酸（H4PteGlu）和CO₂，以及10-HCO-H4PteGlu不依赖NADP⁺水解裂解为H4PteGlu和甲酸。10-FTHFDH在C端有一个485个氨基酸的结构域，与醛脱氢酶（ALDH：EC 1.2.1.3）有46%的同源性，且含有一个保守的活性位点半胱氨酸（Cys-707）。10-FTHFDH催化丙醛的NADP⁺依赖氧化反应以及对硝基苯乙酸（pNPA）的水解反应，其方式与ALDH相似。初始速率研究得出，NADP⁺和丙醛的Km值分别为46和636微摩尔，而pNPA的Km值为220微摩尔。丙醛能够与10-HCO-H4PteGlu竞争NADP⁺依赖的氧化反应，但对不依赖NADP⁺的水解酶反应没有影响。N-乙基马来酰亚胺抑制NADP⁺依赖的10-HCO-H4PteGlu氧化反应，但仅部分抑制（65%）水解酶活性。双硫仑是一种有效的胞质ALDH抑制剂，它抑制10-FTHFDH对NADP⁺依赖的丙醛氧化反应。我们提出，10-FTHFDH的脱氢酶反应机制是通过硫代半缩醛和硫酯中间体进行的，这与醛脱氢酶所描述的机制类似。10-FTHFDH水解酶活性依赖于2-巯基乙醇，可能是检测系统的假象。10-FTHFDH的N端结构域与甘氨酰胺核糖核苷酸转甲酰基酶（EC 2.1.
