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Manipulation of a cation-π sandwich reveals conformational flexibility in phenylalanine hydroxylase
Journal article   Open access   Peer reviewed

Manipulation of a cation-π sandwich reveals conformational flexibility in phenylalanine hydroxylase

Emilia C. Arturo, George W. Merkel, Michael R. Hansen, Sophia Lisowski, Deeanne Almeida, Kushol Gupta and Eileen K. Jaffe
Biochimie, v 183, pp 63-77
Apr 2021
DOI: https://doi.org/10.1016/j.biochi.2020.11.011
PMID: 33221376
Featured in Collection :   UN Sustainable Development Goals @ Drexel
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https://doi.org/10.1016/j.biochi.2020.11.011View
Published, Version of Record (VoR)CC BY-NC-ND V4.0,  Open

Abstract

Allosteric regulation Phenylalanine hydroxylase Phenylketonuria Protein conformation Single residue substitution
Phenylalanine hydroxylase (PAH) is an allosteric enzyme that maintains phenylalanine (Phe) below neurotoxic levels; its failure results in phenylketonuria, an inborn error of amino acid metabolism. Wild type (WT) PAH equilibrates among resting-state (RS-PAH) and activated (A-PAH) conformations, whose equilibrium position depends upon allosteric Phe binding. The RS-PAH conformation of WT rat PAH (rPAH) contains a cation-π sandwich involving Phe80 that cannot exist in the A-PAH conformation. Phe80 variants F80A, F80D, F80L, and F80R were prepared and evaluated using native PAGE, size exclusion chromatography, ion exchange behavior, intrinsic protein fluorescence, enzyme kinetics, and limited proteolysis, each as a function of [Phe]. Like WT rPAH, F80A and F80D show allosteric activation by Phe while F80L and F80R are constitutively active. Maximal activity of all variants suggests relief of a rate-determining conformational change. Limited proteolysis of WT rPAH (minus Phe) reveals facile cleavage within a 4-helix bundle that is buried in the RS-PAH tetramer interface, reflecting dynamic dissociation of that tetramer. This cleavage is not seen for the Phe80 variants, which all show proteolytic hypersensitivity in a linker that repositions during the RS-PAH to A-PAH interchange. Hypersensitivity is corrected by addition of Phe such that all variants become like WT rPAH and achieve the A-PAH conformation. Thus, manipulation of Phe80 perturbs the conformational space sampled by PAH, increasing sampling of on-pathway intermediates in the RS-PAH and A-PAH interchange. The behavior of the Phe80 variants mimics that of disease-associated R68S and suggests a molecular basis for proteolytic susceptibility in PKU-associated human PAH variants. •Phenylalanine hydroxylase variants at Phe80 lack a stabilizing cation-π interaction.•A new and stable undocked phenylalanine hydroxylase conformation is revealed.•PKU-associated phenylalanine hydroxylase variants may favor this conformation.

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Web of Science research areas
Biochemistry & Molecular Biology
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