Mechanism Based Design of Novel Inhibitors of Dinuclear Hydrolases

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This award by the Inorganic, Bioinorganic and Organometallic Chemistry and the Molecular Biochemistry programs supports research by Professor Richard B. Holz at Utah State University to determine a detailed catalytic mechanism for the leucine aminopeptidase from Aeromonas proteolytica (AAP) and the dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) from H. influenzae and to design and syntheize potent inhibitors of DapE. . This research involves an interdisciplinary approach that incorporates biochemical, spectroscopic, and X-ray crystallographic methods and the synthesis of small molecule inhibitors of DapE. The work addresses five key areas: (1) Which residues in the active site of AAP play catalytically important roles? (2) The development of Laue X-ray diffraction methods to study the catalytic mechanism of AAP. (3) Characterization of how substrate- and transition-state analog inhibitors interact with DapE. (4) Preparation of altered enzymes of proposed active site residues in DapE. (5) The development of a detailed mechanism of action for AAP and DapEHydrolases that contain dinuclear metal centers are central to numerous biological processes and, consequently, characterization of their structure and function is a problem of outstanding importance. They play important roles in tissue repair, protein maturation, hormone level regulation, cell-cycle control, as well as cell wall synthesis. Several dinuclear aminopeptidases have broad substrate specificities and are widely distributed in both plant and animal tissues. Their biological and medicinal significance is extensive due to their roles in the degradation of proteins and biologically active peptides, including hormones. The importance of understanding the reaction mechanism of leucine aminopeptidases is underscored by the fact that leucine aminopeptidase activity has been observed on the surface of tumor cells, and such activity is known to play key roles in oncogenesis. In addition, the naturally occurring peptide analog inhibitor, bestatin, significantly decreases HIV infection in males by inhibiting leucine aminopeptidase activity. For DapE's, the emergence of several pathogenic bacterial strains that are resistant to all currently available antibiotics emphasize their importance, since both products of the mDAP/lysine biosynthetic pathway, mDAP and lysine, are essential components of the peptidoglycan cell wall for Gram-negative and most Gram-positive bacteria. This research aims toward a detailed understanding of hydrolytic reactions catalyzed by dinuclear metal centers, which may lead to the development of new antibiotics and also supports the development of undergraduate, graduate and postdoctoral students.