Mammalian zinc metalloendopeptidase EC 3.4.24.15 [EP 24.15] activity is crucial to the formation and degradation of many bioactive peptides. This enzyme has been localized in vivo, and its cDNA cloned and sequenced. The importance of EP 24.15 is demonstrated with the decapeptide gonadotropin releasing hormone [GnRH] (also known as luteinizing hormone releasing hormone), the pivotal neuropeptide regulating mammalian reproduction. Cleavage by EP 24.15, renders GnRH inactive, and is the rate limiting step in its extracellular processing and degradation. EP 24.15 also metabolizes small peptide substrates such as bradykinin, substance P and neurotensin and generates enkephalins from precursor proteins. Recently, this enzyme has also been implicated in the physiology of nociception, blood pressure regulation and pulmonary responsiveness. Therefore, elucidating the function and structure of EP24.15 may yield clues to the pathophysiology of certain diseases, and act as a paradigm for understanding the regulation of neuropeptides and other peptide hormones by a peptidase. Specific objective addressed in this project are: Which critical residues are involved in the enzyme's catalytic mechanisms, active site and substrate specificity? What is responsible for a small substrate preference, and can this be altered? Site-directed mutagenesis and enzyme assays will be performed. Circular dichroism analysis of wild type/mutants as well as intrinsic fluorescence in the presence and absence of the denaturing agent urea will confirm that attenuated activity is not due to changes in global protein conformation (non-native folding). What are the physiological effects of clinically relevant analogues of GnRH on EP 24.15 activity and can a well characterized EP24.15 inhibitor be found, and what is the effect of these EP 24.15 inhibitors upon GnRH degradation? Can structural information in EP 24.15 aid in designing new pharmacologically active agents? The pharmacopoeia defined could be used to develop nonsteroidal managed male and female contraceptives and be used in the treatment of such diverse disorders as sterility, endometriosis, sex-steroid dependent mammary and prostate cancers, and precocious puberty. What is the atomic structure of EP 24.15? How will this information direct future rational drug design? Structural determination will use homologous metalloprotease modelling, X-ray diffraction experiments, simulated annealing analysis and macromolecular simulations of EP 24. 15, inhibitors and GnRH analogues. The realized goals of this research proposal will substantially contribute to the strategy of an integrated approach of biochemical, theoretical, and structural methods to study enzyme-ligand interactions of EP24.15 in neuroendocrinology. While aiding future rational drug design, these results should also be applicable to other macromolecules.
