Research is proposed to study the structure and function relationships of gastric protease pepsin and a homologous enzyme rhizopuspepsin. The goal of the project is to understand the mechanism of catalysis and zymogen activation of aspartic proteases to which pepsin and rhizopuspepsin are basic models. The information derived from the proposed studies is significant in the understanding and medical manipulations of enzymes in the aspartic protease family who are involved in various physiological functions such as protein digestion, intracellular protein turnover, blood pressure regulation, processing of peptide hormone precursor, and others. In previous work, the cDNA clones of pepsinogen and rhizopuspepsin have been identified and sequenced. Future experiments involve site-directed mutagenesis of these proteins in order to test the role of various functional groups. Initial step in this research is the construction of expression vectors for the synthesis of pepsinogen and rhizopuspepsin in E. coli or in mammalian cells. This will be followed by rigorous tests for the integrity of expressed and purified proteins using protein chemistry and enzymic criteria. Kinetic parameters will be determined and binding energies at different steps of enzyme mechanism will be calculated to determine the site of effect by mutations. The choices of the mutagenesis sites will be based on the functional hypothesis as well as the available high-resolution crystal structures. Areas of investigation will include (a) the roles of active site Asp32 and Asp215 in the zymogen activation and in catalysis, (b) the role of hydrogen bonded network at the active site, (c) the role of residues for the catalytic efficiency of long substrates, (d) the role of residues involved in substrate specificity recognition, (e) the roles of residues in the activation peptides of pepsinogen, and (f) the role of phosphoserine at residue 68. Since rhizopuspepsinogen has been identified only from its cDNA structure, the expressed zymogen will also be studied for its properties and activation mechanisms.
