Domain Movements upon Activation of Phenylalanine Hydroxylase Characterized by Crystallography and Chromatography-Coupled Small-Angle X‑ray Scattering

Mammalian phenylalanine hydroxylase
(PheH) is an allosteric enzyme that catalyzes the first step in the
catabolism of the amino acid phenylalanine. Following allosteric activation
by high phenylalanine levels, the enzyme catalyzes the pterin-dependent
conversion of phenylalanine to tyrosine. Inability to control elevated
phenylalanine levels in the blood leads to increased risk of mental
disabilities commonly associated with the inherited metabolic disorder,
phenylketonuria. Although extensively studied, structural changes
associated with allosteric activation in mammalian PheH have been
elusive. Here, we examine the complex allosteric mechanisms of rat
PheH using X-ray crystallography, isothermal titration calorimetry
(ITC), and small-angle X-ray scattering (SAXS). We describe crystal
structures of the preactivated state of the PheH tetramer depicting
the regulatory domains docked against the catalytic domains and preventing
substrate binding. Using SAXS, we further describe the domain movements
involved in allosteric activation of PheH in solution and present
the first demonstration of chromatography-coupled SAXS with Evolving
Factor Analysis (EFA), a powerful method for separating scattering
components in a model-independent way. Together, these results support
a model for allostery in PheH in which phenylalanine stabilizes the
dimerization of the regulatory domains and exposes the active site
for substrate binding and other structural changes needed for activity.