Regulation of Phenylalanine Hydroxylase: Conformational Changes Upon Phenylalanine Binding Detected by Hydrogen/Deuterium Exchange and Mass Spectrometry

Abstract

Phenylalanine acts as an allosteric activator of the tetrahydropterin-dependent enzyme phenylalanine hydroxylase. Hydrogen/deuterium exchange monitored by mass spectrometry has been used to gain insight into local conformational changes accompanying activation of rat phenylalanine hydroxylase by phenylalanine. Peptides in the regulatory and catalytic domains that lie in the interface between these two domains show large increases in the extent of deuterium incorporation from solvent in the presence of phenylalanine. In contrast, the effects of phenylalanine on the exchange kinetics of a mutant enzyme lacking the regulatory domain are limited to peptides surrounding the binding site for the amino acid substrate. These results support a model in which the N-terminus of the protein acts as an inhibitory peptide, with phenylalanine binding causing a conformational change in the regulatory domain that alters the interaction between the catalytic and regulatory domains.Phenylalanine hydroxylase (PheH) 1 is a mononuclear non-heme iron containing monooxygenase that catalyzes the hydroxylation of phenylalanine to tyrosine using molecular oxygen and tetrahydrobiopterin (BH 4 ) (1). This is the rate-limiting step in the catabolism of dietary phenylalanine in the liver. A deficiency in PheH causes the genetic disease phenylketonuria, a common metabolic disorder resulting in mental retardation (2). PheH belongs to the aromatic amino acid hydroxylase family, along with tyrosine hydroxylase (TyrH) and tryptophan hydroxylase (3). The latter two enzymes hydroxylate tyrosine and tryptophan in the biosynthetic pathways for the catecholamine neurotransmitters and serotonin, respectively. The three enzymes have similar chemical † This work was supported in part by grants from the NIH (R01 GM47291) and The Welch Foundation (AQ-1245).*Address correspondence to: Paul F. Fitzpatrick, Department of Biochemistry, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr., San Antonio, TX 78229-3900, ph: 210-567-8264; fax: 210-567-8778, fitzpatrick@biochem.uthscsa.edu. 1 Abbreviations used: PheH, phenylalanine hydroxylase; Δ117PheH, rat PheH lacking the N-terminal 117 residues; TyrH, tyrosine hydroxylase; BH 4 , tetrahydrobiopterin; HXMS, hydrogen/deuterium exchange mass spectrometry; NTA, nitriloacetic acid; MS/MS, tandem mass spectrometry. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 April 20. Published in final edited form as:Biochemistry. The structural basis for activation of PheH by phenylalanine is not known. The only available structures of PheH with an amino acid substrate bound are ternary complexes consisting of the catalytic domain of human PheH with BH 4 and 3-(2-thienyl)-L-alanine or L-norleucine (17,18). These structures provide no insight into the changes in the regulatory domain associated with phenylalanine or BH 4 binding. Indeed, the isolated catalytic domain does not require activation by phenylalanine (19). The only crystal structure containing th...

Identification of the Allosteric Site for Phenylalanine in Rat Phenylalanine Hydroxylase

“…NMR Spectroscopy-NMR experiments were carried out at 298 K on a Brüker Avance 700 MHz spectrometer using cryogenically cooled probes equipped with 13 C and 15 N decoupling and pulsed-field gradient coils. All NMR samples were prepared in 50 mM phosphate, 100 mM NaCl, 1 M leupeptin, 1 M pepstatin A, and 5% D 2 O, pH 8.…”

“…Based on these results, activation of PheH by phenylalanine is proposed to involve a conformational change in which the N-terminal residues move away from the active site (12). Activation of PheH by phenylalanine is well established to cause a significant conformational change in the protein (13), readily detectable as an increase in the fluorescence of the enzyme (14) and exposure of a hydrophobic surface (15).…”

Dynamic regulation of phenylalanine hydroxylase

“…Additionally, regulation of the quaternary structure level could open interesting new opportunities for drug design [46]. The morpheein model explains the drastic changes in local dynamics upon Phe incubation, as observed by H/D exchange mass spectrometry in a hinge region between the regulatory and the catalytic domain including Trp-120 [47]. This region is also susceptible to limited proteolysis, highlighting its flexibility in the Phe-bound state [48].…”

Scallop phenylalanine hydroxylase implicates in immune response and can be induced by human TNF-α

“…There were an ACT domain and a Biopterin_H domain in the deduced CfPAH protein. The ACT domain was an N-terminal regulatory domain to promote allosteric effect, and the Biopterin_H domain was a C-terminal catalytic domain responsible for the conversion from phenylalanine to tyrosine [5,7]. In the Biopterin_H domain of CfPAH, there was a conserved Biopterin-dependent aromatic amino acid hydroxylases signature sequence (from Pro287 to Pro298) with two histidines to bind an iron atom through the form of 2-histidine-1-carboxylate triad [9].…”

“…The regulatory CAT domains share low sequence identity in different animals, and it is devoid in procaryotes such as soil bacterium Chromobacterium violaceum [5,6]. The conformation of the regulatory CAT domain will change after the binding of phenylalanine to alter the interaction between the regulatory and catalytic domain and promote the hydroxylation reaction [7,8]. The homologous catalytic Biopterin_H domains contain an iron-bound motif and a tetramerization motif, and all the residues are required for the determination of substrate specificity and catalytic function [9e11].…”