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.
Highlights
Phenylalanine hydroxylase is one of three enzymes constituting the pterin-dependent amino acid hydroxylase superfamily, all of which catalyze rate-limiting reactions of several metabolic pathways [1]
The steady state fluorescence of wild-type human PAH (hPAH) as well as tryptophan substitution mutants were characterized by Knappskog and Haavik [8], and their spectroscopic changes upon incubation with L-Phe, tetrahydrobiopterin, and Fe(II) have been reported
Activity Measurements—The activity of hPAH was measured after urea or GdnHCl denaturation, performed in the presence or absence of its amino acid substrate
Summary
Materials—Guanidine hydrochloride (GdnHCl), potassium iodide, sodium chloride, sodium thiosulfate, EDTA, and ammonium Fe(II) sulfate were from Merck. (6R)-Tetrahydrobiopterin was purchased from Schircks Laboratories (Switzerland). Enzymatic Activity—PAH activity was measured as described by Martinez et al [16], using an assay mixture containing 1 mM L-Phe, 0.1 M Na-Hepes, pH 7.5, 0.2 M NaCl, 1 mg mlϪ1 catalase, 0 – 8 M urea, 2.0 g of enzyme, and 5 mM DTT, 100 M (NH4)2Fe(II)SO4 and 75 M (6R)tetrahydrobiopterin in a final reaction volume of 100 l and stopped with an equal amount of 1% (v/v) acetic acid in ethanol. The enzyme samples were incubated from 10 min to 3 days at 40 g/ml at room temperature (21 °C) in 100 mM Na-Hepes, pH 7.50, containing 200 mM NaCl, 1.25 mM EDTA, either with or without 0.5 mM L-Phe and from 0 – 8 M urea and/or 0 –3 M GdnHCl. Addition of 10 mM DTT was used to test for disulfide crosslinking. Sigma Plot (Jandel Scientific Corp.) was used for fitting the three-state model to the observed data
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
