MECHANISM OF ENZYME INHIBITION BY PHARMACOLOGICAL AGENTS

描述：哺乳动物磷酸二酯酶（PDE）水解环 在cAMP介导的信号调节中是必需的 转导 它们构成了一大群同工酶， 分类为家族或类型。 显示不同的PDE类型 特征性的组织分布、独特的生化特性和 独特的抑制剂特性。 这些特征解释了生理学上的 这些酶的性质，并直接相关的PDE的功效 抑制剂作为治疗剂。 不同PDE类型的序列比对 显示了一些可能反映催化剂要求的保护 活动 特定的区域和特定的残基， 然而，对于每种PDE类型的独特性质还不知道。 我们以前设计了一个酵母表达系统，用于分析哺乳动物 PDE和这些酶的耐药突变体的遗传选择。 缺失和点突变用于鉴定区域和残基 这是活性和药物结合所必需的。 我们现在建议审查 几种PDE类型中的同工酶特异性，使用改进的方法 这是建立在我们重组酵母系统的优势之上的。 我们将 利用嵌合PDE酶来确定哪些残基是足够的， 在PDE的背景下，赋予独特的生物化学性质， 每种PDE类型的抑制剂敏感性。 这些数据将用于 优化耐药突变体的选择， 鉴定说明这些特性的特定残留物。 还提出了实验来检查底物特异性。 结果 将解决是否同工酶特异性决定因素是本地化的， 在不同形式的偏微分方程中保持不变的模式。 此外该 从这些研究中获得的信息应该对以PDE为中心的 同工酶之间的区别是至关重要的。 它也可以服务于 可作为其它复杂同工酶家族分析的模型。

DESCRIPTION: Mammalian phosphodiesterases (PDEs) hydrolyze cyclic nucleotides and are essential in the regulation of cAMP-mediated signal transduction. They constitute a large group of isozymes that can be categorized into families or types. Different PDE types display characteristic tissue distributions, distinctive biochemical properties and unique inhibitor profiles. These features account for the physiological properties of these enzymes and are directly relevant to the efficacy of PDE inhibitors as therapeutic agents. Sequence alignment of different PDE types shows some conservation that is likely to reflect requirements for catalytic activity. The specific regions, and the particular residues, responsible for the distinctive properties of each PDE type are not yet known, however. We previously devised a yeast expression system for analysis of mammalian PDEs and for genetic selection of drug resistant mutants of these enzymes. Deletions and point mutations were used to identify regions and residues necessary for activity and for drug binding. We now propose to examine isozyme specificity in several PDE types, making use of a modified approach that builds on the strengths of our reconstituted yeast system. We will utilize chimeric PDE enzymes to determine which residues are sufficient, within the context of a PDE, to confer the unique biochemical properties and inhibitor susceptibility of each PDE type. These data will be used to optimize the selection of drug-resistant mutants leading to the identification of specific residues that account for these properties. Experiments to examine substrate specificity are also proposed. The results will address whether isozyme specificity determinants are localized in a pattern that is conserved across divergent forms of PDEs. In addition, the information derived from these studies should be useful for PDE-centered therapeutics where distinction among isozymes is crucial. It may also serve as a model for the analysis of other complex isozyme families.