Yang X M, MacKenzie R E
Department of Biochemistry, McGill University, Montréal, Québec, Canada.
Biochemistry. 1993 Oct 19;32(41):11118-23. doi: 10.1021/bi00092a022.
The recombinant human bifunctional NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase is unique in its absolute requirement for Mg2+ and inorganic phosphate. Both ions affect the affinity of the enzyme for NAD and have no effect on the binding of methylenetetrahydrofolate. The NAD cofactor can be replaced by NADP with a much higher KM and lower VMAX. Kinetic investigation using NADP supports the role of Mg2+ in dinucleotide binding and illustrates that the 2'-phosphate can substitute for phosphate in this process. The human NAD-dependent bifunctional enzyme has a 44% amino acid sequence identity with the dehydrogenase-cyclohydrolase domain of the yeast mitochondrial NADP-dependent trifunctional enzyme encoded by the MIS1 gene, compared to 37% identity with the corresponding domain of the cytosolic trifunctional enzyme. The sequence comparison and the kinetic properties suggest that the NAD bifunctional enzyme is the mammalian homolog of the yeast mitochondrial trifunctional enzyme, which has evolved a unique use of inorganic phosphate to change its dinucleotide specificity from NADP to NAD. Its role is proposed to be in providing formyltetrahydrofolate for the synthesis of formylmethionyl transfer RNA required for the initiation of protein synthesis in mitochondria.
重组人双功能NAD依赖型亚甲基四氢叶酸脱氢酶-亚甲基四氢叶酸环化水解酶的独特之处在于其对Mg2+和无机磷酸的绝对需求。这两种离子都会影响该酶对NAD的亲和力，而对亚甲基四氢叶酸的结合没有影响。NAD辅因子可以被NADP替代，但此时KM值更高，VMAX值更低。使用NADP进行的动力学研究支持了Mg2+在二核苷酸结合中的作用，并表明2'-磷酸可以在此过程中替代磷酸。与由MIS1基因编码的酵母线粒体NADP依赖型三功能酶的脱氢酶-环化水解酶结构域相比，人NAD依赖型双功能酶具有44%的氨基酸序列同一性，而与胞质三功能酶的相应结构域的同一性为37%。序列比较和动力学性质表明，NAD双功能酶是酵母线粒体三功能酶的哺乳动物同源物，它进化出了一种独特的利用无机磷酸的方式，将其二核苷酸特异性从NADP改变为NAD。其作用被认为是为线粒体中蛋白质合成起始所需的甲酰甲硫氨酰转移RNA的合成提供甲酰四氢叶酸。
