Regulation of folate metabolism

描述(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，并研究一种碳代谢的调节。这项研究将着重研究催化蛋氨酸生物合成/再生最后一步的两种酶--钴胺依赖的蛋氨酸合成酶(METH)和钴胺非依赖性蛋氨酸合成酶(METE)的催化机理。这两种酶都催化甲基四氢叶酸的甲基转移到同型半胱氨酸以产生蛋氨酸。人类没有甲氧西林，所以这种关键酶的抑制剂具有潜在的治疗价值。我们的工作将集中在通过脉冲追逐和停流动力学测量来阐明甲基四氢叶酸被激活以取代甲基的机理。我们对冰毒的研究将集中在钴胺辅因子和三种底物甲基四氢叶酸、腺苷蛋氨酸和同型半胱氨酸之间催化甲基转移所需的构象变化。这些研究将结合使用定点突变来不利于选定的构象，以及在存在或不存在底物的情况下进行光谱测量，以辨别突变对酶光谱性质的影响。我们将研究的第三种酶是人亚甲基四氢叶酸还原酶(MTHFR)，它催化形成甲基四氢叶酸。我们最近了解到人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核苷酸生物合成和甲硫氨酸和腺苷甲硫氨酸甲基化之间的分配中起着重要的作用。我们预测，磷酸化将在调节一碳单元的通量方面发挥重要作用。

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.