Rios-Orlandi E M, MacKenzie R E
Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
J Biol Chem. 1988 Apr 5;263(10):4662-7.
The bifunctional NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase from ascites tumor cells has very different kinetic properties from the larger NADP-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase present in all mammalian cells. The NAD-dependent dehydrogenase is unique in that it requires formation of a magnesium.enzyme complex to allow addition of the first substrate, NAD+. It catalyzes an equilibrium ordered kinetic mechanism that has methylenetetrahydrofolate as the last reactant to add and NADH as the last product released. The NADP-dependent dehydrogenase has the same order of addition of substrates, but NADPH is released prior to methenyltetrahydrofolate. The dehydrogenase-cyclohydrolase activities of both enzymes channel methenyltetrahydropteroylglutamate intermediates with the same efficiency which is unaffected by the number of glutamyl residues in the methylenetetrahydrofolate substrate. However, the cyclohydrolase activity of the bifunctional protein is kinetically independent of its dehydrogenase activity, as supported by its lack of inhibition by NAD+, whereas NADP+ strongly inhibits that of the NADP-dependent enzyme. This difference is further demonstrated by the observation that conversion of formyltetrahydrofolate to methylenetetrahydrofolate in the presence of reduced pyridine nucleotide is catalyzed readily only by the bifunctional enzyme.
来自腹水肿瘤细胞的双功能NAD依赖型亚甲基四氢叶酸脱氢酶-亚甲基四氢叶酸环水解酶，与所有哺乳动物细胞中存在的较大的NADP依赖型亚甲基四氢叶酸脱氢酶-亚甲基四氢叶酸环水解酶-甲酰四氢叶酸合成酶具有非常不同的动力学性质。NAD依赖型脱氢酶的独特之处在于，它需要形成镁-酶复合物才能添加第一种底物NAD⁺。它催化一种平衡有序的动力学机制，其中亚甲基四氢叶酸是最后添加的反应物，NADH是最后释放的产物。NADP依赖型脱氢酶的底物添加顺序相同，但NADPH在亚甲基四氢叶酸之前释放。两种酶的脱氢酶-环水解酶活性以相同的效率引导亚甲基四氢蝶酰谷氨酸中间体，这不受亚甲基四氢叶酸底物中谷氨酰残基数量的影响。然而，双功能蛋白的环水解酶活性在动力学上与其脱氢酶活性无关，这一点得到了NAD⁺对其缺乏抑制作用的支持，而NADP⁺则强烈抑制NADP依赖型酶的环水解酶活性。在存在还原型吡啶核苷酸的情况下，仅双功能酶能轻易催化甲酰四氢叶酸向亚甲基四氢叶酸的转化，这一观察结果进一步证明了这种差异。
