Regulation of folate metabolism

描述（由申请人提供）：本研究的长期目标是阐明以四氢叶酸衍生物为辅因子的酶的催化机制，并研究一碳代谢的调控。本研究将重点研究催化蛋氨酸生物合成/再生最后一步的两种酶——钴胺依赖性蛋氨酸合成酶（MetH）和钴胺非依赖性蛋氨酸合成酶（MetE）的催化机制。这两种酶都催化甲基从甲基四氢叶酸转移到同型半胱氨酸以产生蛋氨酸。人类没有MetE，因此这种必需酶的抑制剂具有潜在的治疗价值。我们的工作将集中于阐明甲基四氢叶酸被激活以取代甲基的机制，使用脉冲追踪和停止流动动力学测量。我们对甲基甲基苯丙胺的研究将集中在催化钴胺素辅助因子与三种底物甲基四氢叶酸、腺苷蛋氨酸和同型半胱氨酸之间甲基转移所需的构象变化上。这些研究将采用位点定向诱变的组合，在存在或不存在底物的情况下，对选定的构象和光谱测量不利，以辨别突变对酶的光谱特性的影响。我们将研究的第三种酶是人亚甲基四氢叶酸还原酶（MTHFR），它催化甲基四氢叶酸的形成。我们最近了解到人类MTHFR是磷酸化的，现在希望确定磷酸化对酶活性、亚细胞定位和活性全酶表达的意义。MTHFR在控制一个碳单元在用于核苷酸生物合成和结合到蛋氨酸和腺苷蛋氨酸甲基之间的分配中起重要作用。我们预测磷酸化将在调节单碳单元的通量中发挥重要作用。

DESCRIPTION (provided by applicant): The long-term goals of this research are to elucidate the catalytic mechanisms of enzymes that use tetrahydrofolate derivatives as cofactors, and to study the regulation of one carbon metabolism. This research will emphasize studies on the catalytic mechanisms of two enzymes that catalyze the final step in methionine biosynthesis/regeneration, cobalamin-dependent methionine synthase (MetH) and cobalaminindependent methionine synthase (MetE). Both enzymes catalyze the transfer of a methyl group from methyltetrahydrofolate to homocysteine to produce methionine. Humans do not have MetE, and so inhibitors of this essential enzyme have potential therapeutic value. Our efforts will focus on elucidating the mechanism by which methyltetrahydrofolate is activated for displacement of the methyl group, using pulse-chase and stopped flow kinetic measurements. Our studies of MetH will focus on the conformational changes required to catalyze methyl transfers between the cobalamin cofactor and the three substrates methyltetrahydrofolate, adenosylmethionine, and homocysteine. These studies will employ a combination of site-directed mutagenesis to disfavor selected conformations and spectroscopic measurements in the presence or absence of substrates to discern the effect of mutations on the spectral properties of the enzyme. The third enzyme we will study is human methylenetetrahydrofolate reductase (MTHFR), which catalyzes the formation of methyltetrahydrofolate. We have recently learned that human MTHFR is phosphorylated, and now wish to determine the significance of phosphorylation for enzyme activity, subcellular localization, and expression of active holoenzyme. MTHFR plays an important role in controlling the partitioning of one carbon units between use for nucleotide biosynthesis and incorporation into the methyl group of methionine and adenosylmethionine. We predict that phosphorylation will play an important role in modulating the flux of one-carbon units.