Urea-induced denaturation of human phenylalanine hydroxylase.


Abstract

Human phenylalanine hydroxylase was expressed and purified from Escherichia coli as a fusion protein with maltose-binding protein. After removal of the fusion partner, the effects of increasing urea concentrations on enzyme activity, aggregation, unfolding, and refolding were examined. At pH 7.50, purified human phenylalanine hydroxylase is transiently activated in the presence of 0-4 M urea but slowly inactivated at higher denaturant concentrations. Intrinsic tryptophan fluorescence spectroscopy showed that the enzyme is denatured through at least two distinct transitions. The presence of phenylalanine (L-Phe) shifts the transition midpoint of the first transition from 1.4 to 2.7 M urea, whereas the second transition is unaffected by this substrate. Apparently the free energy of denaturation was almost identical for the free enzyme and for the enzyme-substrate complex, but significant differences in dDeltaG(D)/d[urea] (m(D) values) were observed for the first denaturation transition. In the absence of substrate, a high rate of non-covalent aggregation was observed for the enzyme in the presence of 1-4 M urea. All three tryptophan residues in the enzyme (Trp-120, Trp-187, and Trp-326) were mutated to phenylalanine, either as single mutations or in combination, in order to identify the residues involved in the spectroscopic transitions. A gradual dissociation of the native tetrameric enzyme to increasingly denatured dimeric and monomeric forms was demonstrated by size exclusion chromatography in the presence of denaturants. Study holds ProTherm entries: 6381, 6382, 6383, 6384 Extra Details: additive : EDTA(1.25 mM),transition 1 maltose-binding protein; enzyme activity; free energy;,phenylalanine

Submission Details

ID: zpk8b7Yh3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:31 p.m.

Version: 1

Publication Details
Kleppe R;Uhlemann K;Knappskog PM;Haavik J,J. Biol. Chem. (1999) Urea-induced denaturation of human phenylalanine hydroxylase. PMID:10559199
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