L-phenylalanine binding and domain organization in human phenylalanine hydroxylase: a differential scanning calorimetry study.


Abstract

Human phenylalanine hydroxylase (hPAH) is a tetrameric enzyme that catalyzes the hydroxylation of L-phenylalanine (L-Phe) to L-tyrosine; a dysfunction of this enzyme causes phenylketonuria. Each subunit in hPAH contains an N-terminal regulatory domain (Ser2-Ser110), a catalytic domain (Asp112-Arg410), and an oligomerization domain (Ser411-Lys452) including dimerization and tetramerization motifs. Two partially overlapping transitions are seen in differential scanning calorimetry (DSC) thermograms for wild-type hPAH in 0.1 M Na-Hepes buffer, 0.1 M NaCl, pH 7.0. Although these transitions are irreversible, studies on their scan-rate dependence support that the equilibrium thermodynamics analysis is permissible in this case. Comparison with the DSC thermograms for truncated forms of the enzyme, studies on the protein and L-Phe concentration effects on the transitions, and structure-energetic calculations based on a modeled structure support that the thermal denaturation of hPAH occurs in three stages: (i) unfolding of the four regulatory domains, which is responsible for the low-temperature calorimetric transition; (ii) unfolding of two (out of the four) catalytic domains, which is responsible for the high-temperature transition; and (iii) irreversible protein denaturation, which is likely responsible for the observed exothermic distortion in the high-temperature side of the high-temperature transition. Stages 1 and 2 do not appear to be two-state processes. We present an approach to the analysis of ligand effects on DSC transition temperatures, which is based on the general binding polynomial formalism and is not restricted to two-state transitions. Application of this approach to the L-Phe effect on the DSC thermograms for hPAH suggests that (i) there are no binding sites for L-Phe in the regulatory domains; therefore, contrary to the common belief, the activation of PAH by L-Phe seems to be the result of its homotropic cooperative binding to the active sites. (ii) The regulatory domain appears to be involved in cooperativity through its interactions with the catalytic and oligomerization domains; thus, upon regulatory domain unfolding, the cooperativity in the binding of L-Phe to the catalytic domains seems to be lost and the value of the L-Phe concentration corresponding to half-saturation is increased. Overall, our results contribute to the understanding of the conformational stability and the substrate-induced cooperative activation of this important enzyme. Study holds ProTherm entries: 15250, 15251 Extra Details: Transition 1 tetrameric enzyme; regulatory domain; high-temperature transition; cooperative activation

Submission Details

ID: Jetx2WiU4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:46 p.m.

Version: 1

Publication Details
Thórólfsson M;Ibarra-Molero B;Fojan P;Petersen SB;Sanchez-Ruiz JM;Martínez A,Biochemistry (2002) L-phenylalanine binding and domain organization in human phenylalanine hydroxylase: a differential scanning calorimetry study. PMID:12056888
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1J8U 2002-05-22 1.5 Catalytic Domain of Human Phenylalanine Hydroxylase Fe(II) in Complex with Tetrahydrobiopterin
6HPO 2019-06-05 1.67 Crystallographic structure of the catalytic domain of Human Phenylalanine Hydroxylase (hPAH CD) in complex with iron at 1.6 Angstrom
1J8T 2002-05-22 1.7 Catalytic Domain of Human Phenylalanine Hydroxylase Fe(II)
5FII 2016-03-30 1.8 Structure of a human aspartate kinase, chorismate mutase and TyrA domain.
4PAH 1999-04-27 2.0 HUMAN PHENYLALANINE HYDROXYLASE CATALYTIC DOMAIN DIMER WITH BOUND NOR-ADRENALINE INHIBITOR
1PAH 1999-01-13 2.0 HUMAN PHENYLALANINE HYDROXYLASE DIMER, RESIDUES 117-424
1DMW 2000-03-24 2.0 CRYSTAL STRUCTURE OF DOUBLE TRUNCATED HUMAN PHENYLALANINE HYDROXYLASE WITH BOUND 7,8-DIHYDRO-L-BIOPTERIN
1MMT 2003-09-04 2.0 Crystal structure of ternary complex of the catalytic domain of human phenylalanine hydroxylase (Fe(II)) complexed with tetrahydrobiopterin and norleucine
1MMK 2003-09-04 2.0 Crystal structure of ternary complex of the catalytic domain of human phenylalanine hydroxylase ((FeII)) complexed with tetrahydrobiopterin and thienylalanine
3PAH 1999-04-27 2.0 HUMAN PHENYLALANINE HYDROXYLASE CATALYTIC DOMAIN DIMER WITH BOUND ADRENALINE INHIBITOR
1TDW 2004-11-30 2.1 Crystal structure of double truncated human phenylalanine hydroxylase BH4-responsive PKU mutant A313T.
5PAH 1999-04-27 2.1 HUMAN PHENYLALANINE HYDROXYLASE CATALYTIC DOMAIN DIMER WITH BOUND DOPAMINE INHIBITOR
4ANP 2012-04-11 2.11 Crystal structure of human phenylalanine hydroxylase in complex with a pharmacological chaperone
6PAH 1999-04-27 2.15 HUMAN PHENYLALANINE HYDROXYLASE CATALYTIC DOMAIN DIMER WITH BOUND L-DOPA (3,4-DIHYDROXYPHENYLALANINE) INHIBITOR
1PHZ 1999-04-30 2.2 STRUCTURE OF PHOSPHORYLATED PHENYLALANINE HYDROXYLASE
1TG2 2004-11-30 2.2 Crystal structure of phenylalanine hydroxylase A313T mutant with 7,8-dihydrobiopterin bound
5EGQ 2016-05-18 2.5 Structure of tetrameric rat phenylalanine hydroxylase mutant R270K, residues 25-453
1KW0 2003-01-28 2.5 Catalytic Domain of Human Phenylalanine Hydroxylase (Fe(II)) in Complex with Tetrahydrobiopterin and Thienylalanine
2PHM 1999-04-30 2.6 STRUCTURE OF PHENYLALANINE HYDROXYLASE DEPHOSPHORYLATED
5DEN 2016-02-17 2.9 The First Structure of a Full-Length Mammalian Phenylalanine Hydroxylase Reveals the Architecture of an Auto-inhibited Tetramer
6N1K 2019-05-22 3.06 Full-length human phenylalanine hydroxylase (PAH) in the resting state
2PAH 1999-10-06 3.1 TETRAMERIC HUMAN PHENYLALANINE HYDROXYLASE
6HYC 2019-06-05 3.18 The structure of full-length human phenylalanine hydroxylase in complex with the cofactor and negative regulator tetrahydrobiopterin
5FGJ 2016-05-18 3.6 Structure of tetrameric rat phenylalanine hydroxylase, residues 1-453

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
95.5 Phenylalanine-4-hydroxylase P04176 PH4H_RAT
96.1 Phenylalanine-4-hydroxylase P16331 PH4H_MOUSE
95.8 Phenylalanine-4-hydroxylase Q2KIH7 PH4H_BOVIN
100.0 Phenylalanine-4-hydroxylase P00439 PH4H_HUMAN