Substrate Recognition and Specificity in Thimet Oligopeptidase

Our goal is to understand the substrate recognition and cleavage site specificity in neuropeptidases using thimet oligo peptidase as a model system. Neuropeptidases metabolize and modify the activities of peptide neurotransmitters and neurohormones. Major substrates of thimet oligopeptidase include bradykinin and neurotensin. Bradykinin is a known vasodilator, while neurotensin is also thought to act as a hypotensive agent. Inhibitors of thimet oligopeptidase are, therefore, potential therapeutics for cardiovascular diseases related to hypertension. Efforts to target this enzyme and other neuropeptidases are hampered by our poor understanding of substrate recognition and specificity. Thimet oligopeptidase hydrolyzes only short peptides with a surprisingly diverse range of cleavage site sequences, characteristic features of a number of neuropeptidases. We will use a combination of structural and functional approaches to understand this unusual substrate recognition. We propose that plasticity in the active site is responsible for binding different cleavage site sequences and that a few residues near the active site playa critical role in substrate specificity. To test these hypotheses, we propose to (1) determine crystal structure of thimet oligopeptidase, (2) determine co-crystal structures of thimet oligopeptidase bound with substrate analogues and (3) demonstrate our understanding of substrate recognition by reengineering thimet oligopeptidase to have the specificity of a closely related neuropeptidase. In broad sense, this work will lead to better inhibitor design and therapeutics development targeting thimet oligopeptidase and other neuropeptidases.