The goals of this research are to better understand the mechanism, specificity and biological function of several medically important and unique classes of zinc and/or iron metalloenzymes. (1) Protein farnesyltransferase and protein geranylgeranyltransferase I catalyze the prenylation of many proteins in important signal transduction pathways. We propose to investigate the structure of the catalytic transition state by measuring kinetic isotope effects and the determinants of substrate specificity and product dissociation by mutagenesis. (2) Proteins modified by the prenylation pathways will be identified by first assaying the affinity and catalytic activity of libraries of peptides derived from the human genome and then verified as prenylation targets in vivo using modified prenyldiphosphate substrates and antibody detection. Furthermore, immunoprecipitation and proteomic analysis will be used to interrogate native expression of prenylated proteins. Identification of substrates of this pathway is an important step toward understanding the biological functions of these modifications and the downstream targets of the chemotherapeutic inhibitors of this pathway that are currently in clinical trials. (3) The enzyme UDP-3-0-(R-3-hydroxymyristoyl)-N- acetylglucosamine deacetylase (LpxC) catalyzes the committed step in the synthesis of Lipid A in gram negative bacteria, making LpxC an antibacterial target. We propose to characterize the catalytic mechanism and metal specificity (Fe or Zn) using structure variation together with kinetic and thermodynamic studies. These data will facilitate the design of potent LpxC inhibitors as novel antibiotics especially especially against those organisms associated with cystic fibrosis and some of the potential bioterror agents listed as Ml AID category A and B priority pathogens. (4) Histone deacetylases catalyze the deacetylation of acetylated lysine residues involved in the regulation of gene expression and cell differentiation; inhibitors of HDACs are currently in clinical trials as anticancer drugs. We propose to investigate the identity of the catalytic metal in vivo; the catalytic mechanism; and the substrate specificity, using both known substrates and proteomic approaches. Altering the active site metal may be an important regulatory mechanism of metal-dependent deacetylases. The information gained from these experiments will aid our understanding of the biological function of these important post-translational modifications and enhance the development of novel inhibitors. ? ? ?
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