Our goal is to understand substrate recognition, catalytic function, and the action of inhibitors in neuropeptidases, enzymes that inactivate or modify peptide neurotransmitters or neurohormones. We propose to study specificity and function using two related neuropeptidases, neurolysin and thimet oligopeptidase, as model systems. These zinc metallopeptidases inactivate the neuropeptide neurotensin and are potential therapeutic targets for psychotic disorders and treatment of pain. They have an unusual property shared by a number of neuropeptidases. The cleavage sites they recognize on bioactive peptides are unusually diverse, with no apparent common features. The basis for this unusual property and other aspects of enzyme function and inhibition will be explored by combining high-resolution structure determination with functional studies of the enzymes. Based on prior work, we hypothesize that broad substrate recognition occurs primarily through the interaction between the C termini of substrate peptides and an unusual binding surface, which allows for a variety of different peptide conformations and binding contacts. We also suggest that a hinge-like conformational change in the enzymes accompanies catalysis and that an unusual inhibitor, C28, disrupts enzyme function by preventing this motion.
Four specific aims are proposed: 1) to determine crystal structures of enzymes complexed with peptides and peptide analogs in order to visualize the structural basis for broad specificity, 2) to test and refine our models of recognition by modulating the specificity of the enzyme through mutagenesis, 3) to examine the proposed mechanism of C28 inhibition using velocity sedimentation analysis and other assays 4) to extend our studies to puromycin sensitive aminopeptidase in order to assess the generality of recognition mechanisms. Relevance to public health. The proposed research will provide an understanding of enzymes that are involved in controlling communication between cells in the nervous system. Knowledge of these enzymes will allow us to manipulate their activity in order to treat diseases of the nervous system as well as other disorders
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