l-Phenylalanine Binding and Domain Organization in Human Phenylalanine Hydroxylase:  A Differential Scanning Calorimetry Study

Abstract

Human phenylalanine hydroxylase (hPAH) is a tetrameric enzyme that catalyzes the hydroxylation of L-phenylalanine (L-Phe) to L-tyrosine; a dysfunction of this enzyme causes phenylketonuria. Each subunit in hPAH contains an N-terminal regulatory domain (Ser2-Ser110), a catalytic domain (Asp112-Arg410), and an oligomerization domain (Ser411-Lys452) including dimerization and tetramerization motifs. Two partially overlapping transitions are seen in differential scanning calorimetry (DSC) thermograms for wild-type hPAH in 0.1 M Na-Hepes buffer, 0.1 M NaCl, pH 7.0. Although these transitions are irreversible, studies on their scan-rate dependence support that the equilibrium thermodynamics analysis is permissible in this case. Comparison with the DSC thermograms for truncated forms of the enzyme, studies on the protein and L-Phe concentration effects on the transitions, and structure-energetic calculations based on a modeled structure support that the thermal denaturation of hPAH occurs in three stages: (i) unfolding of the four regulatory domains, which is responsible for the low-temperature calorimetric transition; (ii) unfolding of two (out of the four) catalytic domains, which is responsible for the high-temperature transition; and (iii) irreversible protein denaturation, which is likely responsible for the observed exothermic distortion in the high-temperature side of the high-temperature transition. Stages 1 and 2 do not appear to be two-state processes. We present an approach to the analysis of ligand effects on DSC transition temperatures, which is based on the general binding polynomial formalism and is not restricted to two-state transitions. Application of this approach to the L-Phe effect on the DSC thermograms for hPAH suggests that (i) there are no binding sites for L-Phe in the regulatory domains; therefore, contrary to the common belief, the activation of PAH by L-Phe seems to be the result of its homotropic cooperative binding to the active sites. (ii) The regulatory domain appears to be involved in cooperativity through its interactions with the catalytic and oligomerization domains; thus, upon regulatory domain unfolding, the cooperativity in the binding of L-Phe to the catalytic domains seems to be lost and the value of the L-Phe concentration corresponding to half-saturation is increased. Overall, our results contribute to the understanding of the conformational stability and the substrate-induced cooperative activation of this important enzyme.

Ensemble Perspective for Catalytic Promiscuity

“…Similar results demonstrating resolved transitions for separate structural domains have been noted for the thermal unfolding of phenylalanine hydroxylase, which were attributed to the unfolding of the N-terminal regulatory domain, followed by unfolding of the rest of the enzyme (43). The C-terminal helix region of GSTA1-1 is likewise known to behave in an independent manner compared with the remainder of the protein, and it is likely that the low temperature transition in the DSC thermogram of GSTA1-1 corresponds to changes in this element.…”

Probing the Role of Crystallographically Defined/Predicted Hinge-bending Regions in the Substrate-induced Global Conformational Transition and Catalytic Activation of Human Phenylalanine Hydroxylase by Single-site Mutagenesis

“…3). The observed effect of L-Phe binding on the biphasic thermal denaturation profile of wthPAH (37) (Fig. 3) further supports the interpretation of the SPR response in terms of a biphasic time course, possibly contributed by the two domains (8).…”

“…3). Previous thermal denaturation studies by differential scanning calorimetry and far-UV CD spectroscopy on the wild-type and truncated forms of hPAH (37) have suggested that the thermodynamically irreversible denaturation of the full-length wild-type tetramer occurs through a sequential unfolding of the N-terminal regulatory domains (T m ϳ46°C) and the catalytic domains (T m ϳ54°C) (37). Both transitions are shifted to higher temperatures in the presence of L-Phe (37) (Fig.…”

Irreversible Thermal Denaturation of Glucose Oxidase from Aspergillus niger Is the Transition to the Denatured State with Residual Structure

“…Generally, an increase in the scan rate leads to an increase in the transition temperature up to a so-called high scan limit, where the irreversible step does not affect the hot side of the thermal peak. At this limit, the transition temperature versus the scan rate reaches a plateau (41). Due to the intrinsic limitation of the scan rates used in DSC, the plateau was experimentally unachievable in our measurements (Fig.…”