Stability and folding studies of phenylalanine hydroxylase from Chromobacterium violaceum

Aristobulo Loiza, Purdue University

Abstract

Kinetics of thermal unfolding of apo- and holo-Chromobacterium violaceum phenylalanine hydroxylase (cPAH) has been investigated by circular dichroism (CD) in the temperature range 44 – 76 °C. In addition to the native cofactor (Fe^II), unfolding kinetics of holo-cPAH has been characterized using Zn^II and Co ^II as cofactors. The kinetic profiles for apo- and holo-cPAH show a single-phase exponential rise in CD signal at λ = 222 nm and first-order dependence on protein concentration. The extrapolated unfolding rate constants (k_u) at ambient temperature follow the order apo > Fe > Zn >> Co. Transition-state analysis of the activation parameters reveal an isokinetic correlation, which suggests a shared mechanism for the enzyme variants. The entropy of activation values (ΔS^‡) for apo- and Fe-cPAH are negative but small, -34 ± 24 and -32 ± 18 J mol^-1 K^-1, respectively. On the other hand, ΔS ^‡ values for Zn- and Co-cPAH are 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. Unfolding of holo-cPAH does not show rate dependence on excess metal concentrations and maintains single-phase kinetic profiles, which refute adventitious metal binding and that unfolding occurs via apocPAH exclusively. Isothermal titration calorimetry (ITC) was employed to measure cPAH binding affinities for Fe, Zn, and Co, as well enthalpy of metal coordination. Dissociation constants (K_d) decrease in the order Co < Zn < Fe. The non-native metals, Zn and Co, bind tighter than Fe. The activation enthalpy (ΔH^‡) of unfolding correlates with the enthalpy (ΔH) of metal binding obtained from ITC measurements. 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 the enzyme variants with different metal cofactors. ^ We have investigated the equilibrium (un)folding apo and holo-cPAH using guanidine HCl. Our equilibrium folding studies are the first “folding” studies reported on PAH which reveal information on (1) the elucidation of the folding mechanism at the catalytic domain, (2) the effects that metals play on said mechanism. Unfolding reactions were equilibrated and analyzed by spectroscopic and biophysical methods. CD analysis at λ = 222nm shows two state unfolding transition curves for holo cPAH and three state unfolding curves for apo cPAH with a populated intermediate at 2.2-3M guanidine. Thermodynamic parameters obtained from fitting the unfolding curves to two and three state models show a decrease in ΔG_H2O for holo cPAH along the series that Co²⁺>Fe²⁺ and a global ΔG _H2O of 24.4±6 kJ mol^-1 for apo-cPAH, the majority of the stability coming from apocPAH^I (19 kJmol^-1). The m values of holocPAH suggest a more extended final denatured state for Co-cPAH. Analysis of apo-cPAH^I by DLS yields a Rs = 40 nm and AU data shows a clear species at s20,w = 18.8S, not seen in holo cPAH. Altogether, our data argues against a molten globule as the true identity of apocPAH ^I, but rather argues in favor of a oligomeric species. ^

Degree

Ph.D.

Advisors

Mahdi M. Abu-Omar, Purdue University.

Subject Area

Chemistry, Physical|Biophysics, General

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