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Kinetics of thermal unfolding of phenylalanine hydroxylase variants containing different metal cofactors (FeII, CoII, and ZnII) and their isokinetic relationship.

Publication ,  Journal Article
Loaiza, A; Armstrong, KM; Baker, BM; Abu-Omar, MM
Published in: Inorganic chemistry
June 2008

The kinetics of thermal unfolding of apo- and holo-Chromobacterium violaceum phenylalanine hydroxylase (cPAH) was investigated using circular dichroism (CD) over the temperature range 44-76 degrees C. In addition to the native cofactor (FeII), the unfolding kinetics of holo-cPAH was characterized using ZnII and CoII as cofactors. Kinetic profiles for apo- and holo-cPAH showed a single-phase exponential rise in the CD signal at lambda=222 nm and a first-order dependence on protein concentration. The extrapolated unfolding rate constants (ku) at ambient temperature followed the order apo>Fe>Zn>>Co. Transition-state analysis of the activation parameters revealed an isokinetic correlation, which suggests a common mechanism for the enzyme variants. The values of the entropy of activation (DeltaS++) for apo- and Fe-cPAH were negative but small: -34+/-24 and -32+/-18 J mol(-1) K(-1), respectively. On the other hand, DeltaS++ values for Zn- and Co-cPAH were large and positive: 54+/-9 and 175+/-27 J mol(-1) K(-1), respectively. Therefore, at higher temperatures the unfolding rates of Zn- and Co-cPAH are affected significantly by entropy, while the unfolding rates of apo- and Fe-cPAH are dominated by enthalpy even at higher temperatures. The rate of unfolding of holo-cPAH did not depend on excess metal concentrations and maintained single-phase kinetic profiles, refuting the occurrence of adventitious metal binding and the notion that unfolding occurs via apo-cPAH exclusively. Isothermal titration calorimetry (ITC) was employed to measure cPAH binding affinities for Fe, Zn, and Co as well as the enthalpy of metal coordination. Dissociation constants (Kd) decreased in the order Fe>Zn>Co. The non-native metals, Zn and Co, were bound more tightly than Fe. The activation enthalpy for unfolding (DeltaH++) displayed a linear correlation with the enthalpy of metal binding obtained from ITC measurements (DeltaHITC). On this basis, a common mechanism (transition state) is suggested for this family of metal cofactors, and the varying enthalpy of activation arises from the differing stabilities of enzyme variants having different metal cofactors.

Duke Scholars

Published In

Inorganic chemistry

DOI

EISSN

1520-510X

ISSN

0020-1669

Publication Date

June 2008

Volume

47

Issue

11

Start / End Page

4877 / 4883

Related Subject Headings

  • Temperature
  • Protein Folding
  • Protein Denaturation
  • Protein Conformation
  • Phenylalanine Hydroxylase
  • Models, Molecular
  • Metals, Heavy
  • Kinetics
  • Inorganic & Nuclear Chemistry
  • Enzyme Activation
 

Citation

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Loaiza, A., Armstrong, K. M., Baker, B. M., & Abu-Omar, M. M. (2008). Kinetics of thermal unfolding of phenylalanine hydroxylase variants containing different metal cofactors (FeII, CoII, and ZnII) and their isokinetic relationship. Inorganic Chemistry, 47(11), 4877–4883. https://doi.org/10.1021/ic800181q
Loaiza, Aristobulo, Kathryn M. Armstrong, Brian M. Baker, and Mahdi M. Abu-Omar. “Kinetics of thermal unfolding of phenylalanine hydroxylase variants containing different metal cofactors (FeII, CoII, and ZnII) and their isokinetic relationship.Inorganic Chemistry 47, no. 11 (June 2008): 4877–83. https://doi.org/10.1021/ic800181q.
Loaiza, Aristobulo, et al. “Kinetics of thermal unfolding of phenylalanine hydroxylase variants containing different metal cofactors (FeII, CoII, and ZnII) and their isokinetic relationship.Inorganic Chemistry, vol. 47, no. 11, June 2008, pp. 4877–83. Epmc, doi:10.1021/ic800181q.
Journal cover image

Published In

Inorganic chemistry

DOI

EISSN

1520-510X

ISSN

0020-1669

Publication Date

June 2008

Volume

47

Issue

11

Start / End Page

4877 / 4883

Related Subject Headings

  • Temperature
  • Protein Folding
  • Protein Denaturation
  • Protein Conformation
  • Phenylalanine Hydroxylase
  • Models, Molecular
  • Metals, Heavy
  • Kinetics
  • Inorganic & Nuclear Chemistry
  • Enzyme Activation