XAFS STUDIES OF INTERMEDIATES IN ZINC PROTEASE CATALYSIS

酶机制的直接验证需要了解酶的作用机制。 催化中间体的结构 金属酶类 羧肽酶A在这方面提供了独特机会，因为 金属参与催化作用，金属结合的结构 可以通过X射线吸收精细结构研究溶液中的位置， XAFS分析。 在实验上，同时测量两者 这些中间体的结构和动力学特征需要 延长其非常短的寿命，例如通过降低其速率 的形成和分解。 这可以通过执行 低温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 基本目标，即反应的检测和表征 中间体而不改变反应途径。 特别 这些研究的重要性在于：1）检测结构的方法 变化，2）在零度以下温度下快速测定酶的能力 和3）使用低温溶剂。 必须考虑这些特征中的每一个 仔细研究所考虑的酶。 对于CPD A三个突出 特点是活性需要锌，反应活性高， 对N-末端封闭肽和缩肽的作用及其溶解性 在盐溶液中。 前两个特征都可以作为 产生合适的发色探针。 用Co代替Zn， 在酶的活性位点上的发色原子，其对 结构变化和使用荧光丹磺酰底物阻断 基团可以作为无辐射能量转移测定的基础， 酶活性 这些探针可以帮助鉴定底物， 中间体可以通过冷冻淬灭或平衡来稳定 在零度以下的温度下捕获，这样它们的结构就可以 由XAFS测定。 在平行实验中，HPLC分析将确定 如果在冷冻淬火的中间体中剪切键断裂。 XAFS 允许金属结合位点的结构比较， 用于溶液的底物或抑制剂以及两者的结晶形式 ZnCPD和CoCPD。 CPD A的中间体的结构将是： 与对于具有不同性质的其它锌蛋白酶获得的那些相比， 锌结合位点。 这些活性中心金属的结构 配位络合物在分配电子 在Co可见吸收、CD和MCD光谱中发生的跃迁， 整个建筑群的几何形状的变化， 一个单一的配体。

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.