Our work is directed toward obtaining a fundamental understanding of the basis of serpin interaction with serine proteases. The particular serpin we have chosen to study in detail is alpha1-antichymotrypsin (ACT). ACT is a relatively specific inhibitory of chymotrypsin-like serine proteases [chymotrypsin (Chtr), cathepsin G (CatG)]. In contrast, the serpin alpha1- protease inhibitor (alpha1-PI) is relatively non-specific, being a potent inhibitor of Chtr, trypsin, and elastase (Travis and Salvesen, 1983). In the experiments described below we seek to gain a full understanding of the interaction of ACT with Chtr and CatG, and to test our understanding by redesigning ACT, through site-specific mutagenesis, to change its specificity. First, we will carry out site-specific mutagenesis and chemical modification in order to identify amino acid residues in ACT that are important for its interaction with Chtr (CatG). We will seek answers to the following questions: a) how much of the reactive loop of ACT is covered on forming a complex with Chtr (CatG)/; b) what residues within the reactive loop are most important for interaction with Chtr (CatG), and precisely how do they affect the interaction?; c) how much of the protein is required in order for it to act as an inhibitor?; d) are residues outside the reactive loop important for interaction with Chtr (CatG)? Second, we will make use of site-specific mutagenesis to introduce reporter groups into the reactive loop, and employ stopped-flow spectrophotometry to detect rapidly formed intermediates in the interaction of ACT (and ACT variants) with Chtr (CatG) and determine rate constants for their appearance and disappearance. Third, we will identify which new sites of ACT are cleaved during the newly described """"""""locking"""""""" step in the interaction of ACT and Chtr and determine whether suitable substitution at these sites can significantly alter the mechanism of ACT action. Fourth, we will use site-specific mutagenesis to ask whether by substitution of the active loop of alpha1-PI for the active loop of ACT, and suitable further variation within the loop but leaving the rest of the ACT molecule unchanged, we can convert ACT into an effective inhibitor of human neutrophil elastase (HNE). Fifth, we will use calorimetric methods to the overall equilibrium constants and heats of enthalpy for ACT and ACT variants with Chtr (CatG). Sixth, we will determine the new N-termini produced on reaction of the L358R variant of ACT with Chtr to address the question of the significance of the site of cleavage within the reactive loop for antiprotease activity. Seventh, we will attempt to grow crystals of ACT and of the ACT complex with Chtr suitable for X-ray diffractional analysis at high resolution. Such analysis will be carried out if such crystals are grown.

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National Institute on Aging (NIA)
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Pathobiochemistry Study Section (PBC)
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University of Pennsylvania
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