MECHANISM OF A KEY ENZYME IN AMINO ACID BIOSYNTHESIS

描述：(申请人摘要)详细审查 天冬氨酸-β-半醛酶的结构/功能关系 脱氢酶(ASADH)被提出。这种酶在体内起着关键作用。 生物合成途径导致氨基酸赖氨酸，异亮氨酸， 蛋氨酸和苏氨酸，以及细菌细胞壁的必需成分 生物合成。这项拟议的研究项目的具体目的是 (1)确定天冬氨酸-β-半醛的详细化学机理 脱氢酶，以及(2)确定催化络合物的结构 天冬氨酸-β-半醛脱氢酶。主要的工具将是 应用于这项任务的是定点突变、动力学和同位素 检查酶功能的效果研究，以及X射线结晶学和 分子模拟研究以确定有意义的结构 酶-底物、产物和中间体复合体。该计划涉及 扩大潜在目标氨基酸的名单，以取代 诱变、动力学和同位素效应研究以检测酶的功能， 和X射线结晶学。分子模型研究将用于 确定重要酶的结构-底物、产物和 中间复合体。对ASD基因序列进行了比较 与催化类似反应的其他酶的同源性，包括 具有良好特性的甘油醛-3-磷酸脱氢酶，以及 其他物种的天冬氨酸-β-半醛脱氢酶。区域 具有保守氨基酸序列的将更有可能包含 对酶功能很重要的氨基酸残基，并具有 为我们的诱变研究提供了重要的候选基因。最近的 分子的三维结构的测定 天冬氨酸-β-半醛脱氢酶首席研究员和 他的合作者提供了一些限制条件，以供判断和 完善了许多关于这种酶的机制假说。这一结构 信息还表明，潜在目标的范围扩大了 诱变研究以进一步调查和完善拟议的 机制，鉴定底物结合基团，探索构象 与酶催化有关的变化，并检查 子单元关联的详细信息。

DESCRIPTION: (applicant's abstract) A detailed examination of the structure/function relationships in the enzyme aspartate-beta-semialdehyde dehydrogenase (ASADH) is proposed. This enzyme plays a key role in the biosynthetic pathway leading to the amino acids lysine, isoleucine, methionine and threonine, and to essential components in bacterial cell wall biosynthesis. The specific aims of this proposed research project are to (1) determine the detailed chemical mechanism of aspartate-beta-semialdehyde dehydrogenase, and to (2) determine the structure of the catalytic complexes of aspartate-beta-semialdehyde dehydrogenase. The primary tools that will be applied to this task are site-directed mutagenesis, kinetic and isotope effect studies to examine enzyme function, and x-ray crystallography and molecular modeling studies to determine the structure of significant enzyme-substrate, product, and intermediate complexes. The plan involves expanding the list of potential target amino acids for replacement by mutagenesis, kinetic and isotope effect studies to examine enzyme function, and x-ray crystallography. Molecular modeling studies will be employed to determine the structure of significant enzyme-substrate, product, and intermediate complexes. The asd gene sequence has been compared for homology with other enzymes that catalyze similar reactions, including the well characterized enzyme glyceraldehyde-3-phosphate dehydrogenase, and aspartate-beta-semi-aldehyde dehydrogenases from other species. Regions that have conserved amino acid sequences will be more likely to contain amino acid residues that are important for enzyme function, and have provided significant candidates for our mutagenesis studies. The recent determination of the three-dimensional structures of aspartate-beta-semialdehyde dehydrogenase by the Principal Investigator and his collaborators have provided constraints against which to judge and refine many of mechanistic hypotheses for this enzyme. This structural information has also suggested an expanded set of potential targets for mutagenesis studies to further investigate and refine the proposed mechanism, to identify substrate binding groups, to probe the conformation changes that are associated with enzyme catalysis, and to examine the details of subunit association.