The goals of this research are to better understand the structure, function and distribution of zinc metalloproteins. The catalytic mechanisms and essential active site features of three medically important and novel classes of zinc metalloenzymes will be elucidated, including carbonic anhydrases, protein prenyltransferases and zinc-dependent deacetylases. In the prototypical zinc enzyme, carbonic anhdrase II, roles of the direct metal ligands and the surrounding metal site for determining high catalytic activity and metal selectivity will be investigated using mutagenesis and selection techniques. For protein farnesyltransferase and geranylgeranyltransferase I, the following will be investigated: the structure and reactivity of the metal sites using spectrosocopic methods, metal substitution and mutagenesis; the structure of the transition state by measuring primary and secondary isotope effect; and interactions with peptide substrates that enhance peptide affinity and reactivity using altered substrates and mutagenesis. For UDP-3- O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC), the structure of the metal site will be elucidated using ENDOR, NMR and X-ray crystallography; the functional role of key conserved amino acids will be determined by characterizing the catalytic properties of mutants; and the catalytic mechanism will be probed using pH variation, solvent isotope effects and transient kinetics. Finally, the distribution of zinc metalloproteins in the yeast proteome will be probed using a powerful new """"""""functional genomics"""""""" technology. Carbonic anhydrase-based sensors will be developed to rapidly and specifically measure the zinc content of proteins in a high throughput manner. This assay will then be used to identify all of the zinc-binding proteins in a yeast expression library. These methods should identify novel metalloproteins that have a variety of functions, including: metalloenzymes, transcription factors, and metalloregulatory proteins. Together these data will provide information essential for de novo design of metalloenzymes, for comprehending the function of novel zinc enzymes, for interpreting the in vivo roles of metal ions, for understanding zinc metal homeostasis and human zinc deficiency, and for designing active site inhibitors. In particular, inhibitors of protein farnesyltransferase and LpxC deacetylase in the lipid A pathway may be useful as chemotherapeutics (inhibitors are currently in clinical trials) and antibacterial agents, respectively. Novel antibiotics useful for the treatment of Pseudomonas aeruginosa would be particularly beneficial for patients with AIDS or cystic fibrosis.
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