MECHANISMS OF PEPSIN CATALYSIS AND INHIBITION

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DESCRIPTION (Adapted from applicant's abstract): The objectives of this program are to discover the origin of the unique specificities observed in protein cleavage reactions catalyzed by the aspartic proteinase family. Examples of specificity range from broad, for digestive enzymes, to limited, for enzymes that participate in activation of a single prohormone. As all members of this family have similar three-dimensional structures and catalytic mechanisms, design of selective inhibitors must be based on details of the differences within the active sites. By combining the use of synthetic oligopeptide substrates for kinetic studies, site-directed mutagenesis of residues within the active site clefts contributing to binding and specificity, and comparisons within the family, the precise rationale for tight, specific binding will be discovered. Efforts will focus on the following specific aims: 1. Expansion of the study of active site interactions into the prime region of the cleft. 2. Comparison of the structure and properties of human cathepsin E with the other mammalian enzymes, pepsin and cathepsin D. 3. Generation and analysis of chimeric constructs in which N-terminal and C-terminal domains are interchanged to evaluate the effect of long-range electrostatic effects. 4. Evaluation of the relationship between the three-dimensional structure of the Ascaris pepsin inhibitor and the unique specificity of inhibition. Aim 1 will continue the examination of the extended active site cleft by making new mutations in pepsin, cathepsin D, and a new objective, cathepsin E, which will be studied in detail in aim 2 of the new proposal. Aim 3 proposes an expansion of a mutagenesis strategy through the creation of new protein species where the role of the overall electrostatic environment and mixed backgrounds for point mutations will be explored. In aim 4, the intent is to expand investigation at the protein-protein level by obtaining the structure of a specific natural protein inhibitor. The molecular origins of the strong pepsin directed specificity of the Ascaris inhibitor may be found.