Kinetics of Thermal Unfolding of Phenylalanine Hydroxylase Variants Containing Different Metal Cofactors (Fe super(II), Co super(II), and Zn super(II)) and Their Isokinetic Relationship

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. The rate of unfolding of holo-cPAH did not depend on excess metal concentrations and maintained si...

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Bibliographic Details
Published in:Inorganic chemistry 2008-06, Vol.47 (11), p.4877-4883
Main Authors: Loaiza, Aristobulo, Armstrong, Kathryn M, Baker, Brian M, Abu-Omar, Mahdi M
Format: Article
Language:English
Subjects:
Binding
Cobalt
Dichroism
Enthalpy
Iron
Phenylalanine
Titration calorimetry
Zinc
Online Access:Get full text
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Summary: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. 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. The nonnative metals, Zn and Co, were bound more tightly than Fe. On this basis, a common mechanism (transition state) is suggested for this family of metal co-factors, and the varying enthalpy of activation arises from the differing stabilities of enzyme variants having different metal co-factors.
ISSN:0020-1669
DOI:10.1021/ic800181q