Identification of the Allosteric Site for Phenylalanine in Rat Phenylalanine Hydroxylase

Abstract

Liver phenylalanine hydroxylase (PheH) is an allosteric enzyme that requires activation by phenylalanine for full activity. The location of the allosteric site for phenylalanine has not been established. NMR spectroscopy of the isolated regulatory domain (RDPheH(25-117) is the regulatory domain of PheH lacking residues 1-24) of the rat enzyme in the presence of phenylalanine is consistent with formation of a side-by-side ACT dimer. Six residues in RDPheH(25-117) were identified as being in the phenylalanine-binding site on the basis of intermolecular NOEs between unlabeled phenylalanine and isotopically labeled protein. The location of these residues is consistent with two allosteric sites per dimer, with each site containing residues from both monomers. Site-specific variants of five of the residues (E44Q, A47G, L48V, L62V, and H64N) decreased the affinity of RDPheH(25-117) for phenylalanine based on the ability to stabilize the dimer. Incorporation of the A47G, L48V, and H64N mutations into the intact protein increased the concentration of phenylalanine required for activation. The results identify the location of the allosteric site as the interface of the regulatory domain dimer formed in activated PheH. Phenylalanine hydroxylase (PheH)2 catalyzes a key step in phenylalanine catabolism, the hydroxylation of phenylalanine to tyrosine in the liver using tetrahydropterin (BH 4 ) and oxygen. A deficiency in human PheH increases the level of phenylalanine in the blood, resulting in the inherited disease phenylketonuria (PKU) (1). Thus, the activity of PheH must be tightly controlled to maintain appropriate phenylalanine levels. PheH is activated by phenylalanine and inhibited by BH 4 (2, 3). In addition, phosphorylation of PheH at Ser-16 is reported to decrease the concentration of phenylalanine required to activate PheH (4).Mammalian PheH is a homotetramer, and each monomer contains an N-terminal regulatory domain, a central catalytic domain, and a C-terminal tetramerization domain. The other two aromatic amino acid hydroxylases, tyrosine hydroxylase (TyrH) and tryptophan hydroxylase, have similar architectures.The crystal structures of the catalytic domains of all three enzymes show very similar folds and active sites (5-7), consistent with these enzymes sharing a common catalytic mechanism (8). The structures of the regulatory domains of PheH and TyrH show that both contain ACT domains (5, 9, 10), although the two enzymes are regulated differently (2, 11). To date, there is no published structure of a full-length mammalian PheH, or indeed of any eukaryotic aromatic amino acid hydroxylase, in that the available structures are of proteins lacking the N-terminal regulatory domain, much of the C-terminal tetramerization domain, or both. The structure of a dimeric form of rat PheH containing both the catalytic and regulatory domains but lacking the C-terminal 24 residues required for tetramer formation (5) has provided the present model for the structural basis for activation by phenylalanine. In this structure the...

Domain Movements upon Activation of Phenylalanine Hydroxylase Characterized by Crystallography and Chromatography-Coupled Small-Angle X-ray Scattering

“…3). These results are in agreement with the values obtained by AUC 28 as well as a recent NMR study that shows that RDPheH Δ24 forms a canonical side-by-side ACT domain dimer 30 similar to that observed in prephenate dehydratase 25 (Fig. S3A), with the β2 strand of each monomer forming an extended β-sheet across the dimer interface.…”

“…Two equivalent binding sites for L-Phe were identified in this study, each requiring residues from both chains in the ACT dimer to form. 30 Interestingly, in the crystal structures of the inactive PheH determined in the present study, the β2 strand makes contacts with the catalytic domain (Fig. S3B).…”

“…Based on the accumulating evidence in the literature, 21,26,28,30 as well as our SAXS and ITC data, we propose a minimal ligand-binding model for the allosteric regulation of PheH by L-Phe. The model assumes that the regulatory domains exist in equilibrium between undimerized and dimerized states, and equilibrium is shifted toward the dimerized state by the independent binding of L-Phe to two identical sites per ACT dimer (Fig.…”

The regulatory domain of human tryptophan hydroxylase 1 forms a stable dimer

“…Key to this conformational change is formation of an ACT domain dimer by regulatory domains from two subunits[17, 18]. This model is supported by recent studies of the isolated regulatory domain[14, 19, 20] and of the intact protein[13, 18]. TyrH is activated by phosphorylation of Ser40 in its N-terminal regulatory domain and inhibited by catecholamines[10, 21].…”

“…The resting form of PheH is inactive, with the N-terminus of the regulatory domain partially occluding the active site[12]. Phenylalanine binding to an allosteric site in the regulatory domain[13, 14] causes a significant conformational change that opens up the active site[15, 16]. Key to this conformational change is formation of an ACT domain dimer by regulatory domains from two subunits[17, 18].…”