PROTONATION STATES OF CATALYTIC ASP25 AND ASP25' IN 13C LABELED HIV-1 PROTEASE

13C 标记的 HIV-1 蛋白酶中催化 ASP25 和 ASP25 的质子化态

• 批准号: 7954644
• 负责人: STEPHEN B.H. KENT
• 金额: $ 0.42万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-03-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7954644
• 关键词: Accounting; Aminoisobutyric Acids; Aspartate; Aspartic Endopeptidases; Chemicals; Computer Retrieval of Information on Scientific Projects Database; Coupling; Distant; Enzymes; Equilibrium; Funding; Goals; Grant; HIV-1 protease; Hydrogen Bonding; Institution; Kinetics; Label; Ligand Binding; Mechanics; Methods; Motion; Physiologic pulse; Property; Protein Dynamics; Proteins; Research; Research Personnel; Resources; Series; Source; Spin Labels; Surgical Flaps; Tertiary Protein Structure; Thermodynamics; United States National Institutes of Health; analog; base; chemical synthesis; dimer; enzyme activity; ionization; protonation

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Total chemical synthesis was used to prepare a series of unique chemical analogues of HIV-1 protease, where we systematically substituted the residues Gly51, Gly51' at the tips of the mobile 'flaps' (residues 37-61 in each domain of the protein homodimer) with L-Ala, D-Ala or Aib (aminoisobutyric acid) in both symmetric and asymmetric fashion. Such substitutions, although in regions distant from the catalytic aspartates, led in most cases to reduction of catalytic activity. In contrast to this, a 'covalent dimer' with L-Ala51 in one flap and D-Ala51' in another flap has shown native-like enzyme activity. We used continuous-wave and pulse EPR to characterize dynamic properties of flaps in spin-labeled analogues and found that the thermodynamic balance of the closed, semi-open and open ensembles (with respect to flaps) in HIV-1 protease analogues is perturbed in comparison to that of wild-type enzyme.We interpreted our results on the basis of Northrop's ?kinetic isomechanism' for aspartic proteases which employs a low-barrier hydrogen bond (LBHB) as the central part of the concept. We have developed Northrop's concept further to take into account dynamic properties of the protein and the non-equivalence of the flaps in the ligand-bound enzyme. We proposed a mechanical coupling of motions of the flaps and the catalytic residues Asp25 and Asp25'. On a mechanistic level, opening of the flaps corresponds to shortening of the distance between catalytic residues Asp25 and Asp25' thus promoting formation of the LBHB, and vice versa.Our goal is to employ NMR methods to detect and unambiguously prove presence of LBHB. In addition we would like to determine ionization states of catalytic residues for chemical analogues which demonstrate distinctly different protein dynamics and thus attempt to correlate dynamic properties of the enzyme with its chemical mechanism.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 全化学合成用于制备一系列独特的 HIV-1 蛋白酶化学类似物，其中我们以对称和不对称方式系统地用 L-Ala、D-Ala 或 Aib（氨基异丁酸）取代移动“瓣”尖端的残基 Gly51、Gly51'（蛋白质同源二聚体的每个结构域中的残基 37-61）。这种取代虽然在远离催化天冬氨酸的区域，但在大多数情况下导致催化活性降低。与此相反，一个瓣中含有 L-Ala51 且另一瓣中含有 D-Ala51' 的“共价二聚体”显示出类似天然的酶活性。我们使用连续波和脉冲 EPR 来表征自旋标记类似物中瓣的动态特性，并发现 HIV-1 蛋白酶类似物中封闭、半开放和开放整体（相对于瓣）的热力学平衡与野生型酶相比受到干扰。我们根据诺斯罗普天冬氨酸蛋白酶的“动力学等机制”解释了我们的结果 它采用低势垒氢键（LBHB）作为概念的核心部分。我们进一步发展了诺斯罗普的概念，以考虑蛋白质的动态特性和配体结合酶中襟翼的非等价性。我们提出了襟翼运动与催化残基 Asp25 和 Asp25' 的机械耦合。在机械层面上，瓣的打开对应于催化残基 Asp25 和 Asp25' 之间距离的缩短，从而促进 LBHB 的形成，反之亦然。我们的目标是采用 NMR 方法来检测并明确证明 LBHB 的存在。此外，我们希望确定化学类似物的催化残基的电离态，这些化学类似物表现出明显不同的蛋白质动力学，从而尝试将酶的动态特性与其化学机制相关联。