XAFS STUDIES OF INTERMEDIATES IN ZINC PROTEASE CATALYSIS

锌蛋白酶催化中间体的 XAFS 研究

• 批准号: 3307058
• 负责人: DAVID S AULD
• 金额: $ 23.76万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-01 至 1995-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3307058
• 关键词: X ray spectrometry; active sites; carboxypeptidase; chemical kinetics; cold temperature; enzyme activity; enzyme structure; enzyme substrate complex; high performance liquid chromatography; metal complex; zinc

Direct verification of enzyme mechanisms requires knowledge of the structure of intermediates in catalysis. Metalloenzymes like carboxypeptidase A provide a unique opportunity in this regard because the metal is involved in catalysis and the structure of the metal binding site can be investigated in solution by X-ray Absorption Fine Structure, XAFS analysis. Experimentally, concurrent measurements of both structural and kinetic characteristics of such intermediates requires a lengthening of their very short lifetimes, e.g. by reducing their rates of formation and breakdown. This can be achieved by carrying out the reaction at low temperature and using rapid mixing devices. The ideal system is composed of several features all of which have the same fundamental objective i.e. detection and characterization of reaction intermediates without alteration of the reaction pathway. Of particular importance to these studies are the 1) means to detect structural changes, 2) ability to rapidly assay the enzyme at subzero temperatures and 3) crysolvent employed. Each of these features must be considered carefully for the enzyme under consideration. For CPD A three prominent features are its requirement for zinc for activity, its high reactivity toward N-terminal blocked peptides and depsipeptides and its solubility in salt solutions. Both of the former features can serve as a basis for creating suitable chromophoric probes. Substituting Co for Zn places a chromophoric atom at the active site of the enzyme which is sensitive to structural changes and the use of a fluorescent dansyl substrate blocking group can serve as the basis of a radiatioriless energy transfer assay of enzyme activity. These probes can aid in identifying substrates whose intermediates can be stabilized either by freeze quenching or equilibrium trapping at subzero temperatures, so that their structure can be determined by XAFS. In parallel experiments HPLC analysis will determine if the scissle bond is broken in the freeze quenched intermediate. XAFS allows structural comparisons of the metal binding site complexed with substrates or inhibitors for the solution and crystalline forms of both ZnCPD and CoCPD. The structure of the intermediates of CPD A will be compared to those obtained for other zinc proteases which have different zinc binding sites. The structure of these active site metal coordination complexes should be useful in assigning electronic transitions that occur in Co visible absorption, CD and MCD spectra to changes in the geometry of the complex as a whole or perhaps to movement of a single ligand.

直接验证酶机制需要了解 催化中间体的结构。 金属酶如 羧肽酶 A 在这方面提供了独特的机会，因为 金属参与催化和金属结合的结构 可以通过 X 射线吸收精细结构在溶液中研究位点， XAFS 分析。 实验上，同时测量两者 此类中间体的结构和动力学特征需要 延长其非常短的寿命，例如通过降低他们的费率 的形成和分解。 这可以通过执行以下操作来实现 低温反应并使用快速混合装置。 理想 系统由几个具有相同特征的功能组成 基本目标，即反应的检测和表征 不改变反应途径的中间体。 特别是 这些研究的重要性在于 1) 检测结构的方法 变化，2) 在零下温度下快速测定酶的能力 3)使用冷冻溶剂。 必须考虑其中的每一个特征 仔细考虑所考虑的酶。 对于 CPD A 三个突出的 特点是活性需要锌，反应活性高 N端封闭肽和缩酚肽及其溶解度 在盐溶液中。 前两个功能都可以作为基础 创建合适的发色探针。 用 Co 代替 Zn 可以得到 酶活性位点上的发色原子对 结构变化和荧光丹磺酰底物阻断的使用 基团可以作为无辐射能量转移测定的基础 酶活性。 这些探针可以帮助识别底物 中间体可以通过冷冻猝灭或平衡来稳定 在零度以下的温度下捕获，以便它们的结构可以 由 XAFS 确定。 在平行实验中，HPLC 分析将确定 如果冷冻淬灭中间体中的剪断键断裂。 XAFS 允许与复合的金属结合位点的结构比较 两者的溶液和结晶形式的底物或抑制剂 ZnCPD 和 CoCPD。 CPD A的中间体的结构为 与其他锌蛋白酶获得的结果相比，具有不同的 锌结合位点。 这些活性位点金属的结构 协调复合体应该有助于分配电子 Co 可见吸收、CD 和 MCD 光谱中发生的跃迁 综合体整体几何形状的变化或可能是运动的变化 单个配体。