STRUCTURAL STUDIES OF METHYL CYCLE ENZYMES

这项研究项目的主要目的是阐明 S的三维结构和酶机制 腺苷蛋氨酸合成酶与S-腺苷同型半胱氨酸 (ADOHcy)水解酶。这将通过确定和 分析了天然酶的晶体结构以及 这些酶与底物之间的络合物的结构(或 抑制剂)。 因为ADOMet中介的重要作用 反甲基化在调节细胞活动中发挥作用，酶 参与这些反甲基化是有吸引力的靶子 潜在化疗药物的设计。两者都是Adobe Met 合成酶和同型半胱氨酸水解酶似乎是维持 转甲基化反应。在合理设计之前 这些酶的抑制剂，这些结构数据是 必须达到拟议研究的目标。 足够量的ADOMet合成酶 粪产碱菌的ADOHcy水解酶由 合作者G.D.Markham博士和S.Shimizu教授， 分别进行了分析。我们通常可以长出相对较大的六边形 ADOMet合成酶的双锥体晶体(约1.0毫米)。 晶体属六方晶系，空间群为P6222 或P6422，晶胞尺寸a=128.8，c=140.3埃。一 四聚体酶亚基(Mr=42.000,384个氨基酸残基) 是在结晶学不对称单元中。晶体会产生衍射光 使用常规X射线达到约3.0埃分辨率 室温下的发电机。3.5埃分辨率数据 UO_2Cl2衍生物的本征和3.7埃分辨数据 包括异常散射数据在内的数据是通过使用一个区域 北京理工大学生物技术资源中心的探测器X射线衍射仪 弗吉尼亚大学。计算出的差分Patterson图 使用5埃分辨率数据清楚地显示了大小 UO2的位置。寻找另一种好的重原子导数是 正在进行中。ADOHcy水解酶是由六个六聚体组成的 相同的亚基，分子量为48,000道尔顿。水晶与 得到了四方形状和薄板。四角形 定形晶体可用于结晶学研究， 但都稍微太小了一点。生长大晶体的努力是 正在进行中。

The primary goal of this research project is the elucidation of the three-dimensional structures and enzyme mechanisms of S- Adenosylmethionine (AdoMet) synthetase and S-adenosylhomocysteine (AdoHcy) hydrolase. This will be accomplished by determining and analyzing the crystal structures of native enzymes as well as the structures of complexes between these enzymes and substrates (or inhibitors). Because of the important role that AdoMet mediated transmethylations play in regulating cellular events, the enzymes involved in these transmethylations are attractive targets for the design of potential chemotherapeutic agents. Both AdoMet synthetase and AdoHcy hydrolase seems to be key enzymes to maintain the transmethylation reactions. Prior to the rational design of inhibitors for these enzymes, the structural data which are the target of the proposed research must be obtained. Adequate amounts of AdoMet synthetase from Escherichia coli and AdoHcy hydrolase from Alcaligenes faecalis have been provided by collaborators, Dr. G.D. Markham and Professor S. Shimizu, respectively. We can routinely grow relatively large hexagonal bipyramidal crystals (approximately 1.0 mm) of AdoMet synthetase. The crystals belong to the hexagonal system with space group P6222 or P6422 and cell dimensions a=128.8, c=140.3 angstrom. One subunit (Mr=42.000, 384 amino-residues) of the tetrameric enzyme is in a crystallographic asymmetric unit. The crystals diffract to about 3.0 angstrom resolution using a conventional X-ray generator at room temperature. 3.5 angstrom resolution data of native and 3.7 angstrom resolution data of a UO2Cl2 derivative including anomalous scattering data were collected by using an area detector X-ray diffractometer at the Biotechnology Resource of the University of Virginia. A difference Patterson map calculated by using 5 angstrom resolution data clearly shows the major and minor sites of UO2. A search for another good heavy atom derivative is in progress. AdoHcy hydrolase is a hexamer composed of six identical subunits, molecular weight 48,000 daltons. Crystals with tetragonal shapes and thin plates are obtained. The tetragonal shaped crystals could be suitable for the crystallographic studies, but are slightly too small. Efforts to grow the large crystals are in progress.