rdf-syntax-grammar
mods:
  schema location: http://www.loc.gov/mods/v3 http://www.loc.gov/standards/mods/v3/mods-3-2.xsd
  title info:
    title: Mechanistic and structural studies of phenylalanine hydroxylase from Chromobacterium violaceum
  name:
    name part: Panay Escobar, Aram Joel.
    role:
      role term: creator
  name:
    name part: Fitzpatrick, Paul F.
    role:
      role term: thesis advisor.
  name:
    name part: Reinhart, Gregory D.
    role:
      role term: thesis advisor.
  type of resource: text
  genre: bibliography
  origin info:
    place:
      place term: txu
    date issued: 2011
    issuance: monographic
  language:
    language term: eng
  physical description:
    form: remote
    extent: 1 online resource.
  abstract: The phenylalanine hydroxylase from Chromobacterium violaceum (CvPheH) is a non-heme iron monooxygenase that catalyzes the hydroxylation of phenylalanine. This study presents the use of kinetic isotope effects (KIE) as mechanistic probes to compare the reactivity of CvPheH and that of the eukaryotic aromatic amino acid hydroxylases. This study also describes the use of different spectroscopic and kinetic techniques to identify the hydroxylating intermediate for this enzyme and the assignment of the NMR backbone resonances of CvPheH. Kinetic isotope effects on aromatic and benzylic hydroxylation were used to establish that bacterial and eukaryotic phenylalanine hydroxylases have similar reactivity. The observed KIE on aromatic hydroxylation of 1.4 was shown to be a combination of an inverse isotope effect on the hydroxylation of the amino acid and a normal isotope effect on a subsequent step in the reaction. An isotope effect on benzylic hydroxylation of 10 was found for CvPheH. This result establishes the similar reactivity for CvPheH and the eukaryotic aromatic amino acid hydroxylases and suggests the involvement of a common hydroxylating intermediate. Kinetic isotope effects were used to study the hydroxylation of the aliphatic substrate cyclohexylalanine. The Dkcat value with [1,2,2,3,3,4,4,5,5,6,6-²H₁₁]- cyclohexylalanine is unity with wild-type CvPheH, suggesting that chemistry is not ratelimiting with this substrate. The intramolecular isotope effect calculated using [1,2,3,4,5,6-²H₆]-cyclohexylalanine yields a value of 14. This result is evidence for the involvement of a reactive iron species capable of abstracting a hydrogen atom from the aliphatic carbon in cyclohexylalanine. Analysis of the CvPheH reaction using freeze-quench Mössbauer spectroscopy allowed the detection of an Fe(IV) species in the first turnover of the enzyme. Chemical quench and stopped-flow spectrophotometric methods were used to establish the kinetic competency of the Fe(IV) intermediate…
  note: by Aram Joel Panay Escobar.
  note: "Major Subject: Biochemistry"
  note: Title from author supplied metadata (automated record created 2011-11-01 09:04:20).
  note: Electronic resource.
  note: Doctor of Philosophy Texas A&M University 2010 http://hdl.handle.net/1969.1/ETD-TAMU-2010-08-8584
  note: Includes bibliographical references.
  note: Text (Dissertation)
  subject:
    topic: Major biochemistry
  subject:
    topic: ligand binding
  subject:
    topic: Mechanistic studies
  subject:
    topic: NMR backbone assignment
  subject:
    topic: Phenylalanine hydroxylase
  subject:
    topic: Isotope effects
  identifier: http://hdl.handle.net/1969.1/ETD-TAMU-2010-08-8584
  record info:
    record content source: TXA
    record creation date: 111115
    record change date: 20151202144229.0
    record identifier: in00002718367