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Amino Acid Substitution at Phe80 of Mammalian Phenylalanine Hydroxylase Destabilizes Both Resting‐State and Activated Conformations Increasing the Population of Intermediates.
Author(s) -
Arturo Emilia C.,
Merkel George W.,
Borne Elias,
Hansen Michael Riis,
Lisowski Sophia,
Gupta Kushol,
Jaffe Eileen K.
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.06346
Subject(s) - chemistry , allosteric regulation , phenylalanine hydroxylase , phenylalanine , stereochemistry , proteolysis , amino acid , population , protein structure , tetramer , enzyme , biochemistry , demography , sociology
Phenylalanine hydroxylase (PAH) is an allosteric enzyme responsible for maintaining phenylalanine (Phe) below neurotoxic levels; its failure results in phenylketonuria (PKU). PAH equilibrates among long‐lived conformations, including resting‐state (RS‐PAH) and activated (A‐PAH), whose equilibrium position depends upon allosteric Phe binding to the A‐PAH conformation. The RS‐PAH conformation contains a stabilizing cation‐pi sandwich between Phe80, Arg123, and Arg240 (PDB entry 5DEN), which cannot exist in the A‐PAH conformation. Intrinsic protein fluorescence, enzyme kinetic analysis, native PAGE, size exclusion chromatography, limited proteolysis, and behavior on ion exchange resin are reported for F80A, F80D, F80L, and F80R, many as a function of [Phe]. These data indicate that amino acid substitutions at Phe80 destabilizes both the RS‐PAH and A‐PAH conformations so that intermediate, on‐pathway conformations are longer lived. The addition of Phe allows stabilization of the A‐PAH conformation. Kinetic characterization of F80A and F80D reflects allosteric activation while F80L and F80R are constitutively active. The reaction rates of all Phe80 variants suggest relief of a rate determining conformational change present in the wild type protein. Limited proteolysis of WT rPAH in the absence of Phe reveals facile cleavage within a central C‐terminal 4‐helix bundle, reflecting dynamic dissociation of the PAH tetramer. Under these conditions, the Phe80 variants show proteolytic hypersensitity in a linker region that repositions in the RS‐PAH to A‐PAH conformational interchange; this is protected by addition of Phe. We conclude that manipulation of Phe80 dramatically affects the conformational space sampled by PAH, increasing the population of intermediates between RS‐PAH and A‐PAH. Support or Funding Information NIH 5R01‐NS100081; NIH P30 CA006927