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活性之间的不平衡会产生生化级联反应，通常会建立和传播各种疾病的病理生理基础。我们正在研究一类高相对分子质量的血浆和细胞蛋白质，蛇蛋白--丝氨酸蛋白酶抑制剂，其中一些已经进化出以惊人的速度抑制这些蛋白酶的显著特性。特别是，我们对人中性粒细胞弹性蛋白酶(HNE)的调节感兴趣，因为它在炎症中的作用已经得到了充分的证明。我们将集中在HNE与α1-蛋白酶抑制剂(有时称为α1-抗胰蛋白酶)之间的反应，α1-抗胰蛋白酶是该酶最重要的生理学抑制物。蛇类如何抑制其目标酶只有部分了解，但推测涉及一系列精确计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)切割剪切键的初始化学反应，即被蛋白酶酰化的丝氨酸，3)丝氨酸的构象变化，开始发生在酰化步骤之前或之后，导致酶的构象改变。这最后一步导致酶和丝氨酸被困在共价复合体中，阻止了游离酶的持续催化和释放。我们对丝氨酸如何识别、结合并抑制HNE的问题的解决方法是系统地研究反应途径中每一步的基本结构和动力学特征。我们为每一步提出了一个结构模型，并将使用分析工具的组合来测试这些模型，这些工具包括快速动力学技术、化学反应探针-pH和溶剂同位素效应，以及蛋白质构象的X射线结晶学和光谱测定。

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.