Kinetic and structural analysis of active site mutants of monofunctional NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase from Saccharomyces cerevisiae.
Kinetic and structural analysis of active site mutants of monofunctional NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase from Saccharomyces cerevisiae.
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酿酒酵母单功能 NAD 依赖性 5,10-亚甲基四氢叶酸脱氢酶活性位点突变体的动力学和结构分析。
DOI：
10.1021/bi051038x
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Robertus,JonD
中科院分区：
文献类型：
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作者：
Wagner,Wendi;Breksa3rd,AndrewP;Monzingo,ArthurF;Appling,DeanR;Robertus,JonD
5,10-亚甲基四氢叶酸脱氢酶 (MTD) 催化 5,10-亚甲基四氢叶酸可逆氧化为 5,10-亚甲基四氢叶酸。该反应对于提供合成嘌呤和 dTMP 所需氧化态的一碳单元至关重要。对于大多数 MTD，脱氢酶活性与次甲基-THF 环化水解酶活性共存，作为双功能或三功能酶的一部分。酿酒酵母含有单功能 NAD+ 依赖性 5,10-亚甲基四氢叶酸脱氢酶 (yMTD)。进行了动力学、晶体学和诱变研究来鉴定关键残基，以便进一步了解该酶的反应机制及其明显缺乏环水解酶活性。通过动力学同位素实验（VH/VD=3.3）发现氢化物转移对亚甲基四氢叶酸的氧化具有限速作用，并且氢化物转移至NAD+的面选择性被确定为Pro-R（A特异性）。基于先前解析的 yMTD 与结合 NAD 辅因子的结构构建的模型表明，三个保守氨基酸在底物结合或催化中可能发挥作用： Glu121、Cys150 和 Thr151。突变酶的稳态动力学测量表明，Glu121 和 Cys150 对于脱氢酶活性至关重要，而 Thr151 允许进行一些取代。我们的结果与 Glu121 在正确结合叶酸底物方面的关键作用一致；然而，C150 的确切作用尚不清楚。制备单突变体 Thr57Lys 和 Tyr98Gln 以及双突变体 T57K/Y98Q 来测试以下假设：yMTD 中缺乏环水解酶活性是由于双功能 MTD 中发现的保守 Lys/Gln 对的取代。每个突变体保留脱氢酶活性，但未检测到环水解酶活性。
5,10-Methylenetetrahydrofolate dehydrogenase (MTD) catalyzes the reversible oxidation of 5,10-methylenetetrahydrofolate to 5,10-methenyltetrahydrofolate. This reaction is critical for the supply of one-carbon units at the required oxidation states for the synthesis of purines and dTMP. For most MTDs, dehydrogenase activity is co-located with a methenyl-THF cyclohydrolase activity as part of bifunctional or trifunctional enzyme. The yeastSaccharomyces cerevisiaecontains a monofunctional NAD+-dependent 5,10-methylenetetrahydrofolate dehydrogenase (yMTD). Kinetic, crystallographic, and mutagenesis studies were conducted to identify critical residues in order to gain further insight into the reaction mechanism of this enzyme and its apparent lack of cyclohydrolase activity. Hydride transfer was found to be rate-limiting for the oxidation of methylenetetrahydrofolate by kinetic isotope experiments (VH/VD= 3.3), and the facial selectivity of the hydride transfer to NAD+was determined to be Pro-R(A-specific). Model building based on the previously solved structure of yMTD with bound NAD cofactor suggested a possible role for three conserved amino acids in substrate binding or catalysis: Glu121, Cys150, and Thr151. Steady-state kinetic measurements of mutant enzymes demonstrated that Glu121 and Cys150 were essential for dehydrogenase activity, whereas Thr151 allowed some substitution. Our results are consistent with a key role for Glu121 in correctly binding the folate substrate; however, the exact role of C150 is unclear. Single mutants Thr57Lys and Tyr98Gln and double mutant T57K/Y98Q were prepared to test the hypothesis that the lack of cyclohydrolase activity in yMTD was due to the substitution of a conserved Lys/Gln pair found in bifunctional MTDs. Each mutant retained dehydrogenase activity, but no cyclohydrolase activity was detected.