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Title page for ETD etd-12042017-111654


Type of Document Dissertation
Author Mitchener, Michelle Marie
URN etd-12042017-111654
Title A. Competition and allostery govern substrate selectivity of cyclooxygenase-2 B. Enzymatic oxidation of M1dG in the genome
Degree PhD
Department Chemistry
Advisory Committee
Advisor Name Title
Lawrence J. Marnett Committee Chair
Brian O. Bachmann Committee Member
Ned A. Porter Committee Member
Walter J. Chazin Committee Member
Keywords
  • M1dG
  • chemical kinetics
  • endocannabinoids
  • cyclooxygenase-2
  • DNA adducts
Date of Defense 2017-11-16
Availability unrestricted
Abstract
Part A: Cyclooxygenase-2 (COX-2) oxygenates arachidonic acid (AA) and its ester analog, 2-arachidonoylglycerol (2-AG), to prostaglandins (PGs) and prostaglandin glyceryl esters (PG-Gs), respectively. Although the efficiency of oxygenation of these substrates by COX-2 in vitro is similar, cellular biosynthesis of PGs far exceeds that of PG-Gs. Evidence that the homodimeric COX enzymes are functional heterodimers comprising a catalytic and an allosteric subunit suggests that competitive interaction of AA and 2-AG at the allosteric site of COX-2 might result in differential regulation of the oxygenation of the two substrates when both are present. Modulation of AA levels in RAW264.7 macrophages uncovered an inverse correlation between cellular AA levels and PG-G biosynthesis. In vitro kinetic analysis using purified protein demonstrated that the inhibition of 2-AG oxygenation by high concentrations of AA far exceeded the inhibition of AA oxygenation by high concentrations of 2-AG. An unbiased systems-based mechanistic model of the kinetic data revealed that binding of AA or 2-AG at the allosteric site of COX-2 results in a decreased catalytic efficiency of the enzyme toward 2-AG, whereas 2-AG binding at the allosteric site increases COX-2's efficiency toward AA. The results suggest that substrates interact with COX-2 via multiple potential complexes involving binding to both the catalytic and allosteric sites. Competition between AA and 2-AG for these sites, combined with differential allosteric modulation, gives rise to a complex interplay between the substrates, leading to preferential oxygenation of AA.

Part B: Lipid and DNA peroxidation give rise to DNA adducts, the most abundant of which is 3-(2-deoxy-β-D-erythro-pentofuranosyl)pyrimido[1,2-α]purin-10(3H)-one (M1dG). Increased levels of M1dG are positively correlated with a variety of physiological and pathophysiological conditions including aging, bacterial infection, and cancer. Recently, our laboratory reported the oxidative metabolism of M1dG to 6-oxo-M1dG in nuclear DNA isolated from multiple human and rodent cell lines. Heat-denaturation or proteinase K-treatment of nuclear lysates ablated conversion of M1dG to 6-oxo-M1dG in double-stranded oligonucleotides, suggesting that the oxidation is enzymatic. Preincubation of nuclear lysates with iron or alpha-ketoglutarate increased 6-oxo-M1dG formation, and nuclear lysates preincubated with either N-oxalylglycine or IOX1, two structurally distinct broad-spectrum alpha-ketoglutarate-dependent dioxygenase inhibitors, showed reduced 6-oxo-M1dG formation. Finally, treatment of RKO cells with JIB-04, a pan alpha-ketoglutarate-dependent Jumonji histone demethylase inhibitor, resulted in reduced levels of genomic 6-oxo-M1dG along with a concomitant increase in levels of M1dG. Collectively, these findings indicate the presence of a nuclear enzyme capable of converting M1dG to 6-oxo-M1dG in genomic DNA and support the hypothesis that the enzyme is a member of the alpha-ketoglutarate-dependent dioxygenase class of iron-dependent enzymes that is responsible for the oxidation of various alkylated nucleic acids and/or amino acids. An IOX1-based photoaffinity-based probe is currently being used to identify the enzyme(s), and oligonucleotides bearing site-specifically incorporated 6-oxo-M1dG are being generated for the characterization of this adduct’s mutagenic potential.

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