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.
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