This award in the Inorganic, Bioinorganic and Organometallic Chemistry program supports research by Professor Richard C. Holz at the Loyala University of Chicago to develop an understanding of hydrolytic reactions catalyzed by metallohydrolases that contain dinuclear active sites. These enzymes catalyze diverse reactions such as the degradation of DNA, RNA, phospholipids, and polypeptides. The research will explore why some hydrolases utilize a mononuclear center while others function with either a mononuclear or dinuclear active site, and still others require two metal ions to catalyze the same chemical reaction. These systems use different metal ion Lewis acidities in discrete dinuclear sites to i) bind and position substrate, ii) bind and activate a water molecule to yield an active site hydroxide nucleophile, and/or iii) stabilize the transition state of the hydrolytic reaction.
Structurally detailed catalytic mechanisms will be developed for the leucine aminopeptidase from Vibrio (Aeromonas) proteolyticus (AAP), the dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) from H. influenzae, and the argE-encoded N-acetyl-L-ornithine deacetylase (ArgE) E. coli. The experimental approach incorporates biochemical, spectroscopic, X-ray crystallographic methods.and site directed mutants of catalytically important amino acid residues, and small molecule inhibitors. The specific aims are: i) Which metal ion in AAP functions as the catalytic metal ion?, ii) What are the catalytic roles of active site residues that reside in the second coordination sphere?, iii) What residues in the active site of DapE and ArgE play catalytically important roles?, iv) How do substrate- and transition-state analog inhibitors interact with DapE and ArgE?, v) Synthesize novel inhibitors of DapE and ArgE that may function as antibacterial agents.
This research addresses the mechanism of action of dinuclear metalloproteases which play a central role in several disease states including stroke, diabetes, cancer, HIV, bacterial infections, and neuropsychiatric disorders associated with the dysregulation of glutamatergic neurotransmission, such as schizophrenia, seizure disorders, and amyotrophic lateral sclerosis (ALS). This knowledge may lead to the development of coordination complexes that are capable of catalyzing industrially important hydrolytic reactions. In addition, these mechanistic data will aid in the design of small molecules that function as new anti-bacterial agents.
