DYNAMICS OF GLUTATHIONE TRANSFERASES

描述(申请人提供)：本研究的长期目标 程序是确定一种酶机制的分子基础 一类称为谷胱甘肽转移酶的酶。这些蛋白质形成了一个家族 解毒酶通过结合谷胱甘肽到广泛的 各种可能有害的疏水化合物。他们已经被证明 在肿瘤生长的启动过程中起着重要作用 化疗药物耐药性的发展。总而言之，这些 酶表现出非常广泛的底物特性。 这项提案的目标是确定 这些酶的分子动力学和酶功能。X射线衍生品 目前已知有六个类别的结构。尽管总体来说， 这些酶是相似的，每一类都有一个不同的分子 既影响底物专一性又影响酶的结构 机制。对于这些类中的三个类(α、Mu和pi)， 确定了这些蛋白质的X-射线衍生化结构。在某些情况下， 这些结构表明酶的构象发生了相当大的变化。 到配基结合。在其他情况下，底物结合几乎不会引起 结构。因为所有这些研究都是在水晶中进行的 晶格，配体引起结构变化的程度和重要性 这些酶在溶液中的动力学尚不清楚。一个更全面的 对这些酶的了解将有助于开发更有用的 化疗药物。 这项建议的具体目的是研究底物诱导的变化。 用核磁共振研究人类类酶u、pi、α和theta的动态变化 光谱学。第一个要检验的假设是分子动力学 脊椎在这些酶的作用机制中起着重要作用。 酶通过门控底物的可获得性和产物的释放。动态感 存在和不存在时这些酶中主链原子的性质 不同的底物和产品的测量将通过测量 酰胺交换动力学、剩余偶极偶联、化学交换和15N 核松弛。需要检验的第二个假设是，动态 侧链残基的性质在分子识别中起着重要作用 不同的底物由同一种酶。野生型和野生型的侧链动力学 突变的蛋白质将通过‘3C，2H和19F核旋来表征 放松一下。

DESCRIPTION (provided by applicant): The long range goal of this research program is to determine the molecular basis of the enzymatic mechanism of a class of enzymes called glutathione transferases. These proteins form a family of detoxification enzymes that function by conjugating glutathione to a wide variety of potentially harmful hydrophobic compounds. They have been shown to play an important role in the initiation of tumor growth, as well as in the development of resistance to chemotherapeutic drugs. Collectively, these enzymes show a remarkably broad range of substrate specificities. The goal of this proposal is to determine the relationship between the molecular dynamics and enzyme function for these enzymes. The x-ray derived structures of six classes are currently known. Although the overall fold of these enzymes are similar, each class possesses a distinct molecular architecture which affects both the substrate specificity as well as the enzyme mechanism. For three of these classes (alpha, mu, and pi), a large number of x-ray derived structures of these proteins have been determined. In some cases, these structures show considerable change in the conformation of the enzyme due to ligand binding. In other cases, substrate binding causes little change in structure. Since all of these studies have been performed in the crystalline lattice, the extent and importance of ligand induced changes on the structure and dynamics of these enzymes in solution is unknown. A more comprehensive understanding of these enzymes will be useful in the development of more useful chemotherapeutics. The specific aims of this proposal are to investigate substrate induced changes in the dynamics of human class mu, pi, alpha, and theta enzymes by NMR spectroscopy. The first hypothesis to be tested is that molecular dynamics of the backbone plays an important role in the enzymatic mechanism of these enzymes by gating substrate accessibility and product release. The dynamic properties of the backbone atoms in these enzymes in the presence and absence of various substrates and products will be investigated with measurements of amide exchange kinetics, residual dipolar coupling, chemical exchange, and 15N nuclear relaxation. The second hypothesis to be tested is that the dynamic properties of side-chain residues play an important role in the recognition of different substrates by the same enzyme. Side chain dynamics of wild-type and mutant proteins will be characterized by '3C, 2H, and 19F nuclear spin relaxation.