SERINE PROTEASE INHIBITORS & MECHANISMS OF INFLAMATION

该研究计划的总体目标是了解参与炎症和宿主防御的丝氨酸蛋白酶的调节。 丝氨酸蛋白酶活性和抑制之间的不平衡产生生化级联反应，其通常建立和传播多种疾病的病理生理学基础。 我们正在研究一个家族的高分子量血浆和细胞蛋白质，丝氨酸蛋白酶抑制剂-丝氨酸蛋白酶抑制剂，其中一些已经进化出显着的性能，抑制这些蛋白酶以极快的速度。 特别是，我们感兴趣的人中性粒细胞弹性蛋白酶（HNE）的调节，因为它在炎症中的作用有据可查。 我们将重点关注HNE和α 1-蛋白酶抑制剂（有时称为α 1-抗胰蛋白酶）之间的反应，这是生理上最重要的酶抑制剂。 丝氨酸蛋白酶抑制剂如何抑制其靶酶仅部分了解，但推测涉及一系列精确定时的化学和构象步骤。一般的抑制机制有三个关键特征，1）识别和结合事件，2）切割易断裂键的初始化学反应，即，丝氨酸蛋白酶抑制剂被蛋白酶酰化，3）在酰化步骤之前或之后开始发生的丝氨酸蛋白酶抑制剂的构象变化，导致酶构象的改变。 这最后一步导致酶和丝氨酸蛋白酶抑制剂被捕获在共价复合物中，从而防止继续催化和释放游离酶。我们对丝氨酸蛋白酶抑制剂如何识别，结合，然后抑制HNE的问题的方法是系统地检查反应途径中的每一步的基本结构和动力学特征。 我们提出了每个步骤的结构模型，并将测试这些模型使用的分析工具，包括快速动力学技术，化学反应性-pH值和溶剂同位素效应的探针，和X射线晶体学和光谱测定蛋白质构象的组合。

The overall goal of the research program is to understand the regulation of serine proteases involved in inflammation and host defenses. An imbalance between serine protease activity and inhibition generates biochemical cascades that often establish and propagate the pathophysiological basis of a wide variety of diseases. We are studying a family of high molecular weight plasma and cellular proteins, the serpins--serine protease inhibitors, some of which have evolved the remarkable property of inhibiting these proteases with astonishingly fast rates. In particular, we are interested the regulation of human neutrophil elastase (HNE) because of its well documented role in inflammation. We will focus on the reaction between HNE and alpha1-protease inhibitor (sometimes referred to as alpha1- antitrypsin) which is physiologically the most important inhibitor of the enzyme. How serpins inhibit their target enzymes is only partially understood but is presumed to involve a series of exquisitely timed chemical and conformational steps. There are three critical features of the general inhibitory mechanism, 1) the recognition and binding event, 2) the initial chemical reaction in cleaving the scissile bond, i.e., acylation, of the serpin by the protease, 3) a conformational change in the serpin that starts to occur either prior to, or following, the acylation step leading to an alteration of the conformation of the enzyme. This last step results in the enzyme and serpin trapped in a covalent complex that prevents continued catalysis and release of free enzyme. Our approach to the problem of how a serpin recognizes, binds and then inhibits HNE is to systematically examine the basic structural and kinetic features of each step in the reaction pathway. We propose a structural model for each step and will test these models using combinations of analytical tools including rapid kinetic techniques, probes of chemical reactivity--pH and solvent isotope effects, and X-ray crystallographic and spectroscopic determinations of protein conformation.