SALIVARY AMYLASE--CONFORMATION AND DOMAIN STRUCTURE

唾液淀粉酶——构象和结构域

• 批准号: 2131522
• 负责人: NARAYANAN RAMASUBBU
• 金额: $ 9.01万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-30 至 1997-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2131522
• 关键词: Streptococcus; X ray crystallography; amylases; bioassay; chemical binding; circular dichroism; computer program /software; conformation; enzyme activity; fluorescence spectrometry; human subject; hydroxyapatites; molecular dynamics; molecular site; protein denaturation; protein purification; protein structure function; saliva

Salivary amylase provides an excellent example to illustrate the principles that have emerged from the study of salivary function in the oral cavity, namely, the multifunctionality, amphifunctionality, and the conformational dictum of salivary components. The multifunctional nature of amylase includes: 1) hydrolysis of complex carbohydrates; 2) binding to hydroxyapatite (HAP); and 3) binding to bacteria (e,g, streptococci) in solution and when bound to HAP. The amphifunctionality arises from the final outcome of the functions. In other words, the different functions of a single molecule may be beneficial or potentially harmful depending upon the site of action. For salivary amylase, its binding to bacteria in solution may result in bacterial clearance (protective) while its presence in the enamel pellicle may facilitate dental plaque formation at the enamel surface (harmful). Its binding to viridans streptococci both in solution as well as when bound to the HAP surface is dictated by the maintenance of its native conformation. Alpha- amylases consist of three structural domains, A, B and C. Based on the structural homology among alpha-amylases, a hypothesis that the bacterial and substrate binding functions are associated with distinct, conformationally independent structural domains is proposed. As a corollary to this hypothesis, we propose that the C-domain of amylase is responsible for bacterial binding. As part of a long range goal for the design of amylase molecules for use in artificial salivas, this application will obtain critical baseline information using biophysical and biochemical techniques. Protein X-ray crystallography will be used to obtain the 3D structure of amylase. Biophysical and biochemical techniques will be used to define the structural domains associated with the three functions of amylase listed above and to test whether or not the intact C-domain is solely responsible for bacterial binding. Comparison of the biological properties of native amylase and fragments obtained by selective fragmentation or partial denaturation will be used to identify the structural domains that are responsible for its functions. On the basis of the information derived from these studies, molecular modeling and dynamics will be used to differentiate between the substrate and bacterial binding domains and to identify residues suitable in these domains for future mutagenesis experiments.

唾液淀粉酶提供了一个很好的例子来说明 唾液腺功能研究中得出的原理 口腔，即多功能性、两功能性和 唾液成分的构象法则。 多功能性 淀粉酶的作用包括：1）复杂碳水化合物的水解； 2) 绑定 羟基磷灰石（HAP）； 3) 与细菌（例如链球菌）结合 在溶液中以及与 HAP 结合时。 两性功能来源于 函数的最终结果。 换句话说，不同的 单个分子的功能可能有益，也可能有害 取决于作用部位。 对于唾液淀粉酶，其结合 溶液中的细菌可能会导致细菌清除（保护性），而 它存在于牙釉质薄膜中可能会促进牙菌斑的形成 在牙釉质表面形成（有害）。 它与草绿色结合 链球菌在溶液中以及与 HAP 表面结合时 是由其天然构象的维持决定的。 阿尔法- 淀粉酶由三个结构域组成：A、B 和 C。 α-淀粉酶之间的结构同源性，假设细菌 和底物结合功能与不同的相关， 提出了构象独立的结构域。 作为一个 根据这一假设，我们提出淀粉酶的 C 结构域是 负责细菌结合。 作为长期目标的一部分 用于人工唾液的淀粉酶分子的设计，这 应用程序将使用生物物理获取关键基线信息 和生化技术。 将使用蛋白质X射线晶体学 获得淀粉酶的3D结构。 生物物理和生化 技术将用于定义与相关的结构域 上面列出了淀粉酶的三个功能并测试是否 完整的 C 结构域仅负责细菌结合。 天然淀粉酶和片段的生物学特性比较 将使用通过选择性片段化或部分变性获得的 确定对其负责的结构域 功能。 根据这些研究得出的信息， 分子模型和动力学将用于区分 底物和细菌结合域并鉴定合适的残基 在这些领域进行未来的诱变实验。