The phenylketonuria-associated substitution R68S converts phenylalanine hydroxylase to a constitutively active enzyme but reduces its stability

Abstract

The naturally occurring R68S substitution of phenylalanine hydroxylase (PheH) causes phenylketonuria (PKU). However, the molecular basis for how the R68S variant leads to PKU remains unclear. Kinetic characterization of R68S PheH establishes that the enzyme is fully active in the absence of allosteric binding of phenylalanine, in contrast to the WT enzyme. Analytical ultracentrifugation establishes that the isolated regulatory domain of R68S PheH is predominantly monomeric in the absence of phenylalanine and dimerizes in its presence, similar to the regulatory domain of the WT enzyme. Fluorescence and small-angle X-ray scattering analyses establish that the overall conformation of the resting form of R68S PheH is different from that of the WT enzyme. The data are consistent with the substitution disrupting the interface between the catalytic and regulatory domains of the enzyme, shifting the equilibrium between the resting and activated forms ϳ200-fold, so that the resting form of R68S PheH is ϳ70% in the activated conformation. However, R68S PheH loses activity 2 orders of magnitude more rapidly than the WT enzyme at 37°C and is significantly more sensitive to proteolysis. We propose that, even though this substitution converts the enzyme to a constitutively active enzyme, it results in PKU because of the decrease in protein stability. cro ARTICLE

Manipulation of a Cation-π Sandwich Reveals Conformational Flexibility in Phenylalanine Hydroxylase

“…One interpretation of a variety of biochemical and biophysical data is that the R68S variant may be "undocked" such that the entire regulatory domain lacks intra-and intersubunit interaction with the catalytic domain (as in the RS-PAH conformation, see Fig 1B) nor an intersubunit interaction with a neighboring ACT domain (as in the A-PAH conformation, see Fig 1C), thereby increasing solvent accessibility of loops and protein interfaces that are otherwise protected. The undocked interpretation is consistent with high thermal instability for R68S (14), studies showing that this amino acid substitution interferes with allosteric Phe binding (31), and in vivo studies indicating enhanced protein degradation relative to wild type (32,33). In the following description of the Phe80 variants, we find it useful to consider RS-PAH, A-PAH, as well as…”

“…We cannot yet predict how these substitutions impact the A-PAH conformation, where we do not know the molecular interactions of Phe80, as an atomic resolution structure is lacking. We can however predict, based on all A-PAH models, that Phe80 is expected to extend from the inner surface of the ACT-domain dimer towards the rest of the protein (see (14). One interpretation of a variety of biochemical and biophysical data is that the R68S variant may be "undocked" such that the entire regulatory domain lacks intra-and intersubunit interaction with the catalytic domain (as in the RS-PAH conformation, see Fig 1B) nor an intersubunit interaction with a neighboring ACT domain (as in the A-PAH conformation, see Fig 1C), thereby increasing solvent accessibility of loops and protein interfaces that are otherwise protected.…”

“…Prior limited trypsinolysis studies uniformly focused on differential cleavage within a "hinge region" between the regulatory and catalytic domains (residues 111-117) (e.g. (14,44,45)).…”

“…1B (5,11). In addition, there are now a series of publications that address improved modelling of the A-PAH conformation first introduced in 2013, the most recent of which is illustrated in Fig 1C (5,7,(11)(12)(13)(14). However, no crystal structure exists for full-length PAH (rat or human) in the A-PAH conformation, and little experimental attention has been given to the mechanism for the transition between the RS-PAH and A-PAH conformations.…”

“…However, it has long been realized that PAH activation minimally involves releasing autoinhibition through movement of the N-terminal 24 – 30 amino acids (segment 2, which would by necessity drag along segment 1) (29,30). A recent report on a constitutively active rat PAH (rPAH) model of a disease-associated human PAH (hPAH) variant, R68S, suggests the existence of conformations wherein autoinhibition is relieved but ACT domain dimerization is not present, thus uncoupling these phenomena (14). One interpretation of a variety of biochemical and biophysical data is that the R68S variant may be “undocked” such that the entire regulatory domain lacks intra- and inter-subunit interaction with the catalytic domain (as in the RS-PAH conformation, see Fig 1B) nor an intersubunit interaction with a neighboring ACT domain (as in the A-PAH conformation, see Fig 1C), thereby increasing solvent accessibility of loops and protein interfaces that are otherwise protected.…”