PT  - JOURNAL ARTICLE
AU  - Emilia C. Arturo
AU  - George W. Merkel
AU  - Michael R. Hansen
AU  - Sophia Lisowski
AU  - Deeanne Almeida
AU  - Kushol Gupta
AU  - Eileen K. Jaffe
TI  - Manipulation of a Cation-π Sandwich Reveals Conformational Flexibility in Phenylalanine Hydroxylase
AID  - 10.1101/2020.02.28.969873
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.02.28.969873
4099  - http://biorxiv.org/content/early/2020/11/11/2020.02.28.969873.short
4100  - http://biorxiv.org/content/early/2020/11/11/2020.02.28.969873.full
AB  - 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 resting-state (RS-PAH) and activated (A-PAH) conformations, whose equilibrium position depends upon allosteric Phe binding to the A-PAH conformation. The RS-PAH conformation of WT rat PAH (rPAH) contains a cation-π sandwich between Phe80, Arg123, and Arg420, which cannot exist in the A-PAH conformation. Phe80 variants F80A, F80D, F80L, and F80R were prepared; their conformational equilibrium was 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 involving Phe80. Limited proteolysis of WT rPAH in the absence of Phe reveals facile cleavage within a C-terminal 4-helix bundle that is buried in the RS-PAH tetramer interface, reflecting dynamic dissociation of the RS-PAH conformation. 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 conformational interchange. Hypersensitivity is corrected by addition of Phe such that all Phe80 variants become like WT rPAH and achieve the A-PAH conformation. Thus, manipulation of Phe80 perturbs the conformational space sampled by PAH, increasing the propensity to sample intermediates in the RS-PAH and A-PAH interchange, which are presumed on-pathway because they can readily achieve the A-PAH conformation by addition of Phe.Competing Interest StatementThe authors have declared no competing interest.