Tissue Stores of Folate: Dietary Control and Assay

描述(申请人提供)：这项研究试图确定肝脏的一种主要酶，甘氨酸N-甲基转移酶(GNMT)的性质。我们发现大鼠肝脏中的GNMT与5-甲基四氢叶酸五氢谷氨酸酯(5-CH3-THF-Gs)结合并被抑制。这是一项监管计划的一部分，该计划将通过叶酸单碳库从头合成甲基与从蛋氨酸获得甲基联系起来。5-CH3-THF-Gs不抑制重组GNMT在大肠杆菌中的表达。我们发现，5-CH3-THF-G5对天然GNMT的抑制作用依赖于酶抑制剂和底物的加入顺序。我们的第一个假设是，抑制依赖于重组酶中不存在的翻译后修饰。我们的第二个假设是，这种翻译后修饰可能是天然酶的磷酸化。我们的第三个假设是，天然酶和重组酶之间存在结构和构象上的差异。我们的第四个假设是，当酶和底物结合时，酶的构象发生变化。为了验证这些假设，我们的第一个特定目标是将GNMT表达到进行翻译后修饰的人H1299细胞中，并检测5-CH3-THF-Gs对其的抑制和结合。我们的第二个特定目的是确定天然大鼠GNMT的磷酸化位点，并对H1299细胞表达的GNMT进行磷酸化氨基酸的突变，以确定哪些氨基酸需要抑制。天然GNMT是N-末端乙酰化的。我们的第三个具体目标是去除N-末端的乙酰基，以确定这是否是天然酶和重组酶之间差异的来源。我们的第四个特定目标是确定抑制剂5-CH3-THF-Gs与H1299中表达的GNMT结合的位置。我们将通过在没有和有结合抑制剂的情况下解决GNMT的晶体结构来做到这一点。我们还将尝试将5-CH3-THF-G共价连接到GNMT上，并通过胰酶消化和质谱学确定抑制剂的位置。我们的第五个具体目标是测量在底物和抑制剂存在的情况下天然GNMT的构象变化。我们将使用[32P]-磷酸标记GNMT，然后胰酶消化，GNMT的免疫纯化，标记多肽的高效液相分离和Edman测序来定位磷酸氨基酸，以及磷酸氨基酸的质谱学鉴定和任何其他翻译后修饰。构象的变化将通过固有的蛋白质荧光来监测。用超滤法测定抑制剂的结合素9。

DESCRIPTION (provided by applicant): This study seeks to determine properties of a major enzyme of liver, glycine N-methyltransferase (GNMT). We had discovered that GNMT from rat liver binds and is inhibited by 5-methyltetrahydrofolate pentaglutamate (5-CH3-THF-Gs). This is part of a regulatory scheme that links the de novo synthesis of methyl groups via the folate one-carbon pool to the availability of methyl groups from methionine. Recombinant GNMT expressed in E. coil is not inhibited by 5-CH3-THF-Gs. We found that inhibition of native GNMT by 5-CH3-THF-G5 depends upon the order of addition of inhibitor and substrates to the enzyme. Our first hypothesis is that inhibition depends on a post-translational modification not present in the recombinant enzyme. Our second hypothesis is that this post-translational modification may be phosphorylation of the native enzyme. Our third hypothesis is that there are structural and conformational differences between the native and recombinant enzymes. Our fourth hypothesis is that conformational changes in the enzyme take place when enzyme and substrates bind. To test these hypotheses, our first specific aim is to express GNMT transfected into human H1299 cells that carry out post-translational modification and examine inhibition and binding by 5-CH3-THF-Gs. Our second specific aim is to identify the sites of phosphorylation in native rat GNMT, GNMT expressed in H1299 cells and to mutate the phosphorylated amino acids from H1299 cells to determine which are needed for inhibition. Native GNMT is N-terminal acetylated. Our third specific aim is to remove the N-terminal acetyl group to determine whether this the source of the difference between native and recombinant enzyme. Our fourth specific aim is to determine where the inhibitor, 5-CH3-THF-Gs, is bound to the GNMT expressed in H1299. We will do this by solving the crystal structure of GNMT without and with the bound inhibitor. We will also attempt to covalently link 5-CH3-THF-G to GNMT and determine the position of the inhibitor by tryptic digestion and mass spectrometrometry. Our fifth specific aim is to measure conformational changes of native GNMT in the presence of substrates and inhibitor. We will use labeling of GNMT by [32P]-phosphate followed by tryptic digestion, immunopurification of GNMT, HPLC isolation of labeled peptides and Edman sequencing to locate phospho-amino acids as well as mass-spectroscopic identification of phospho-amino acids and any other post-translational modifications. Changes in conformation will be monitored by intrinsic protein fluorescence. Bindin 9 of inhibitor will be measured by ultrafiltration.