Manipulation of a cation-Π sandwich expands knowledge of the conformational flexibility of mammalian phenylalanine hydroxylase

Phenylalanine hydroxylase (PAH) is an allosteric enzyme responsible for maintaining phenylalanine (Phe) below neurotoxic levels; its failure results in phenylketonuria. Wild type (WT) 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 of WT rat PAH (rPAH) contains a stabilizing cation-Π sandwich between Phe80, Arg123, and Arg240, which cannot exist in the A-PAH conformation. We report intrinsic protein fluorescence, enzyme kinetics, native PAGE, size exclusion chromatography, limited proteolysis, and ion exchange behavior for F80A, F80D, F80L, and F80R. These data indicate that amino acid substitution at Phe80 affects both RS-PAH and A-PAH conformations so that intermediate, on-pathway conformations are longer lived. For all variants, Phe addition promotes accumulation of the A-PAH conformation. Kinetic characterization of F80A and F80D shows 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 C-terminal 4-helix bundle, reflecting dynamic dissociation of the PAH tetramer in the RS-PAH conformation. Under these conditions, the Phe80 variants show proteolytic hypersensitity in a linker that repositions in the RS-PAH to A-PAH conformational interchange; this cleavage is protected by addition of Phe. We conclude that manipulation of Phe80 perturbs the conformational space sampled by PAH, increasing the population of conformationally dynamic intermediates in the RS-PAH and A-PAH interchange.