MECHANISMS OF PEPSIN CATALYSIS & INHIBITION

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This project will provide new information on details of interactions between substrate and enzyme at the active site of the Aspartic Proteinase family of enzymes. We believe that the level of kcat for pepsin-like enzymes is controlled through hydrogen bonding to the backbone of peptide groups of the substrates while the level of Km is dominated by interactions with side-chain groups of the substrate. The new information we obtain will be valuable in design of new drugs relevant to control of peptic ulcers (gastric pepsin), hypertension (kidney renin), and prevention of viral maturation of HTLV-III, the virus suspected in the pathophysiology of Acquired Immune Deficiency Syndrome (retroviral protease). Our approach to achieving these ends will take advantage of very new experimental results from our kinetic studies as well as x-ray crystallographic data and cloning and sequence data from other laboratories. ln the previous grant period we have refined our kinetic assay utilizing defined synthetic, chromophoric substrates. From the pH dependence of hydrolysis of these substrates, several new interactions have been revealed. With the availability of site-directed mutagenesis, it is now possible to test our hypotheses about the importance of these interactions in detail by making defined changes in enzyme residues suspected of contributing vital contacts. Collaborations have been arranged with several laboratories that have made progress in cloning the necessary genes or determining the high resolution x-ray structure of key enzymes. The combination of a 1) facile assay, 2) sensitive test for interactions by a study of the pH dependence of kinetics, 3) greatly improved ability to synthesize variants of the substrate structure, 4) complementary ability to obtain selected alterations in enzyme residues, 5) high resolution crystallographic structures, and 6) new generation of ideas about potential interactions in the active site cleft should make this project highly successful.