Krupenko S A, Wagner C, Cook R J
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee.
J Biol Chem. 1995 Jan 13;270(2):519-22. doi: 10.1074/jbc.270.2.519.
The enzyme, 10-formyltetrahydrofolate dehydrogenase (10-FTHFDH) (EC 1.5.1.6) catalyzes both the NADP(+)-dependent oxidation of 10-formyltetrahydrofolate to tetrahydrofolate and CO2 and the NADP(+)-independent hydrolysis of 10-formyltetrahydrofolate to tetrahydrofolate and formate. The COOH-terminal domain of the 10-FTHFDH (residues 417-902) shows a 46% identity with a series of NAD(+)-dependent aldehyde dehydrogenases (EC 1.2.1.3). All known members of the aldehyde dehydrogenase family and 10-FTHFDH have a strictly conserved cysteine (Cys-707 for 10-FTHFDH), which has been predicted to be at the active site of these enzymes. Rat liver 10-FTHFDH was expressed in a baculovirus system, and site-directed mutagenesis has been used to study the role of cysteine 707 in the activity of 10-FTHFDH. 10-FTHFDH with alanine substituted for cysteine at position 707 had no dehydrogenase activity, while hydrolase activity and binding of NADP+ were unchanged. Light scattering analysis revealed that wild type and mutant 10-FTHFDH exist as tetramers. We conclude that cysteine 707 is directly involved in the active site of 10-FTHFDH responsible for dehydrogenase activity, and there is a separate site for the hydrolase activity.
10-甲酰四氢叶酸脱氢酶（10-FTHFDH）（EC 1.5.1.6）催化10-甲酰四氢叶酸依赖NADP(+)氧化为四氢叶酸和二氧化碳，以及10-甲酰四氢叶酸不依赖NADP(+)水解为四氢叶酸和甲酸。10-FTHFDH的COOH末端结构域（第417 - 902位氨基酸残基）与一系列依赖NAD(+)的醛脱氢酶（EC 1.2.1.3）有46%的同源性。醛脱氢酶家族的所有已知成员以及10-FTHFDH都有一个严格保守的半胱氨酸（10-FTHFDH的Cys-707），据预测它位于这些酶的活性位点。大鼠肝脏10-FTHFDH在杆状病毒系统中表达，并且已使用定点诱变来研究半胱氨酸707在10-FTHFDH活性中的作用。在第707位氨基酸残基处用丙氨酸取代半胱氨酸的10-FTHFDH没有脱氢酶活性，而水解酶活性和NADP+的结合未改变。光散射分析表明野生型和突变型10-FTHFDH均以四聚体形式存在。我们得出结论，半胱氨酸707直接参与10-FTHFDH负责脱氢酶活性的活性位点，并且存在一个单独的水解酶活性位点。
