DYNAMICS OF GLUTATHIONE TRANSFERASES

谷胱甘肽转移酶的动力学

基本信息

批准号：
6786677
负责人：
GORDON S. RULE
金额：
$ 21.74万
依托单位：
CARNEGIE-MELLON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-08-01 至 2006-07-31
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：本研究的长期目标程序是确定酶促机制的分子基础一类酶称为谷胱甘肽转移酶。这些蛋白质组成一个家族解毒酶的作用是通过将谷胱甘肽与多种物质结合来发挥作用。各种潜在有害的疏水化合物。他们已被证明在肿瘤生长的启动以及肿瘤生长过程中发挥着重要作用对化疗药物产生耐药性。总的来说，这些酶表现出非常广泛的底物特异性。该提案的目标是确定这些酶的分子动力学和酶功能。 X 射线得出目前已知六类结构。虽然整体折叠这些酶是相似的，每一类都具有不同的分子影响底物特异性和酶的结构机制。对于其中三个类别（alpha、mu 和 pi），大量这些蛋白质的 X 射线衍生结构已被确定。在某些情况下，这些结构显示酶的构象发生了相当大的变化配体结合。在其他情况下，底物结合几乎不会引起结构。由于所有这些研究都是在晶体中进行的晶格，配体诱导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。更全面的了解这些酶将有助于开发更有用的酶化疗。该提案的具体目的是研究底物引起的变化通过 NMR 检测人类 mu、pi、α 和 theta 类酶的动力学光谱学。第一个要检验的假设是分子动力学主链在这些酶促机制中起着重要作用通过门控底物可及性和产物释放来控制酶。动态这些酶存在和不存在时主链原子的特性将通过测量来研究各种基材和产品的酰胺交换动力学、残余偶极耦合、化学交换和 15N 核弛豫。要检验的第二个假设是动态侧链残基的性质在识别中起着重要作用同一种酶作用于不同的底物。野生型和突变蛋白的特征是“3C、2H 和 19F 核自旋” 松弛。