Title page for ETD etd-08182011-160213


Type of Document Dissertation
Author Benson, Brian
Author's Email Address bbenson@lsu.edu
URN etd-08182011-160213
Title Inhibition and Nucleic Acid Binding Studies of the Carboxyltransferase Component of Bacterial Acetyl-CoA Carboxylase
Degree Doctor of Philosophy (Ph.D.)
Department Biological Sciences
Advisory Committee
Advisor Name Title
Waldrop, Grover Committee Chair
Laine, Roger Committee Co-Chair
Grove, Anne Committee Member
Russo, Paul Committee Member
Gimble, Jeffrey Dean's Representative
Keywords
  • enzyme inhibition
  • acetyl-CoA carboxylase
  • DNA-binding protein
Date of Defense 2011-07-12
Availability unrestricted
Abstract
Acetyl-CoA carboxylase is an essential enzyme, as it catalyzes the first committed and regulated step in fatty-acid biosynthesis in all organisms excepting few Archaea and Eubacteria. Acetyl-CoA carboxylase from gram-negative and gram-positive bacteria is a multifunctional enzyme composed of three separate proteins. The carboxyltransferase subunit catalyzes the transfer of a carboxyl group from carboxybiotin to acetyl-CoA, forming malonyl-CoA. The crystal structure of the Escherichia coli (E. coli) carboxyltransferase component of acetyl-CoA carboxylase revealed a unique Zn-domain, presumed to mediate nucleic acid binding, that is absent in the eukaryotic enzyme. Notably, the Zn-domain, adjacent to the active site of carboxyltransferase, makes for a unique target in the development of novel antibiotics capable of highly specific binding. Utilizing an Electrophoretic Mobility Shift Assay as part of this study, we investigated the nonspecific nucleic-acid binding and substrate (malonyl-CoA and biocytin) inhibition of DNA:carboxyltransferase complex formation. Inhibition of carboxyltransferase activity by single-stranded DNA, double-stranded DNA, RNA, and heparin was measured in the reverse direction with a spectrophotometric assay in which the production of acetyl-CoA was coupled with the combined citrate synthase-malate dehydrogenase reaction requiring NAD+ reduction (Blanchard and Waldrop, 1998). NADH formation was followed spectrophotometrically at 340 nm. We then determined and characterized the mechanism of inhibition by tetracycline (and derivatives) on carboxyltransferase from E. coli and Staphylococcus aureus. The tetracyclines are broad-spectrum antibiotics that inhibit translation by binding to the 30S ribosomal subunit and preventing the binding of the acylated-tRNA to the A-site. Tetracycline exhibited competitive inhibition with respect to both malonyl-CoA and biocytin. Multiple inhibition analyses with a bisubstrate analog showed that tetracycline and the substrates can bind to the enzyme simultaneously. Surprisingly, tetracycline did not interfere with the DNA-binding properties of carboxyltransferase. This introduction begins with a historical perspective of carboxylation reactions. Next biotin and the structure, function and practical applications of acetyl-CoA carboxylase are described. Subsequently a review of moonlighting enzymes, or those capable of catalyzing reactions in basic metabolism while acting as regulators of gene expression, is provided, as are the functions and structures of several types of zinc finger.
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