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
1PHZ 1998-11-11T00:00:00+0000 2.2 STRUCTURE OF PHOSPHORYLATED PHENYLALANINE HYDROXYLASE
2PHM 1998-11-11T00:00:00+0000 2.6 STRUCTURE OF PHENYLALANINE HYDROXYLASE DEPHOSPHORYLATED
5DEN 2015-08-25T00:00:00+0000 2.9 The First Structure of a Full-Length Mammalian Phenylalanine Hydroxylase Reveals the Architecture of an Auto-inhibited Tetramer
5EGQ 2015-10-27T00:00:00+0000 2.5 Structure of tetrameric rat phenylalanine hydroxylase mutant R270K, residues 25-453
5FGJ 2015-12-20T00:00:00+0000 3.6 Structure of tetrameric rat phenylalanine hydroxylase, residues 1-453
1DMW 1999-12-15T00:00:00+0000 2.0 CRYSTAL STRUCTURE OF DOUBLE TRUNCATED HUMAN PHENYLALANINE HYDROXYLASE WITH BOUND 7,8-DIHYDRO-L-BIOPTERIN
1J8T 2001-05-22T00:00:00+0000 1.7 Catalytic Domain of Human Phenylalanine Hydroxylase Fe(II)
1J8U 2001-05-22T00:00:00+0000 1.5 Catalytic Domain of Human Phenylalanine Hydroxylase Fe(II) in Complex with Tetrahydrobiopterin
1KW0 2002-01-28T00:00:00+0000 2.5 Catalytic Domain of Human Phenylalanine Hydroxylase (Fe(II)) in Complex with Tetrahydrobiopterin and Thienylalanine
1LRM 2002-05-15T00:00:00+0000 2.1 Crystal structure of binary complex of the catalytic domain of human phenylalanine hydroxylase with dihydrobiopterin (BH2)

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