Panay Escobar, Aram Joel (2010-08). Mechanistic and Structural Studies of Phenylalanine Hydroxylase from Chromobacterium violaceum. Doctoral Dissertation. Thesis uri icon

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 M?ssbauer 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 as the hydroxylating species. The NMR amide backbone resonances in the HSQC spectrum of CvPheH were assigned to the corresponding amino acid residues using a suite of TROSY-based threedimensional triple resonance experiments. We were able to assign 224 residues out of the 278 assignable residues in CvPheH, this constitutes 81 percent of the assignable protein sequence.
  • 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 M?ssbauer 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 as the hydroxylating species.

    The NMR amide backbone resonances in the HSQC spectrum of CvPheH were

    assigned to the corresponding amino acid residues using a suite of TROSY-based threedimensional

    triple resonance experiments. We were able to assign 224 residues out of

    the 278 assignable residues in CvPheH, this constitutes 81 percent of the assignable protein

    sequence.

publication date

  • August 2010