XAFS STUDIES OF INTERMEDIATES IN ZINC PROTEASE CATALYSIS

直接验证酶的机制需要了解 催化中间体的结构。金属酶，如 羧基肽酶A在这方面提供了一个独特的机会，因为 金属参与了催化和金属结合的结构 可以用X射线吸收精细结构研究溶液中的晶位， XAFS分析。在实验上，同时测量这两种方法 这类中间体的结构和动力学特性要求 延长他们非常短的寿命，例如通过降低他们的费率 形成和分解。这可以通过执行 在低温下进行反应，并使用快速混合装置。理想 系统由几个功能组成，所有这些功能都具有相同的功能 基本目标，即反应的检测和表征 在不改变反应路径的情况下合成中间体。特别的 对这些研究的重要性是1)检测结构的手段 变化，2)在零下温度下快速测定酶的能力 3)使用冷冻剂。必须考虑这些特性中的每一个 仔细寻找正在考虑的酶。对于CPD A来说，三个突出的 特点是它的活性需要锌，它的高反应性 N-末端封闭多肽和脱脂多肽及其溶解性 在盐溶液中。前两个功能都可以作为 创造出合适的发色探针。用钴取代锌使a 酶活性部位的发色原子对其敏感 荧光底物丹磺酰封闭剂的结构变化及应用 基团可作为无辐射能量转移分析的基础 酶活性。这些探针可以帮助识别其底物 中间体可以通过冷冻淬火或平衡来稳定。 在零下的温度下捕获，这样它们的结构就可以 由XAFS确定。在平行实验中，高效液相分析将确定 如果冷冻淬火的中间体中的剪刀键断裂。XAFS 允许对金属结合部位的结构进行比较 用于这两者的溶液和晶型的底物或抑制剂 ZnCPD和CoCPD。CPD A中间体的结构将是 与其他锌蛋白酶的结果相比，这些酶具有不同的 锌结合部位。这些活性中心金属结构 配位络合物在分配电子原子时应该是有用的 Co-可见光吸收光谱、Cd光谱和MCD光谱的跃迁 建筑群作为一个整体或可能是运动的几何形状的变化 一个单一的配基。

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.