Direct evidence for a phenylalanine site in the regulatory domain of phenylalanine hydroxylase.

The physiological role of the liver enzyme phenylalanine hydroxylase (PheH)1 is to catalyze the hydroxylation of excess phenylalanine in the diet to form tyrosine, using tetrahydrobiopterin (BH4) as the source of electrons for this monooxygenation reaction (Figure 1) [1]. The importance of the enzyme is demonstrated by the devastating effects of insufficient PheH activity. The resulting disease, phenylketonuria, results in poor growth and progressive intellectual impairment, with eventual death of the affected patient at a young age in the absence of treatment [2]. Figure 1 The reaction catalyzed by phenylalanine hydroxylase PheH is a member of the family of aromatic amino acid hydroxylases, along with tyrosine hydroxylase (TyrH) and tryptophan hydroxylase (TrpH) [1]. TyrH hydroxylase in the adrenal gland and central nervous system catalyzes the rate-limiting step in the formation of catecholamine neurotransmitters. TrpH in the central nervous system catalyzes the rate-limiting step in the formation of serotonin. All three enzymes are homotetramers, with each monomer containing an N-terminal regulatory domain of 100-160 residues, a homologous catalytic domain of ~300 residues, and a C-terminal tetramerization domain of ~45 residues that contains a C-terminal helix. There is no structure available of an intact eukaryotic aromatic amino acid hydroxylase. In the case of PheH, the most complete structures are of a dimeric rat enzyme that lacks the C-terminal helix [3] and of the tetrameric human enzyme lacking the N-terminal regulatory domain [4]. The structure of the dimeric rat enzyme shows that the N-terminus of the regulatory domain extends across the active site opening. This feature has led to residues 19-29 being designated an auto-inhibitory domain critical for regulation of PheH [5]. Maintaining proper levels of PheH activity is clearly critical, and the enzyme activity is regulated in response to levels of free phenylalanine. The seminal work leading to our present understanding of the regulation of PheH was carried out by Shiman and coworkers [6–9]. PheH exhibits positive cooperativity when the rate of the reaction is determined as a function of phenylalanine concentration and hyperbolic kinetics when the concentration of the tetrahydropterin is varied. In addition, there is a lag in the formation of tyrosine unless the enzyme is first incubated with phenylalanine [9], and the lag is more pronounced if the enzyme is first incubated with a tetrahydropterin. In the model developed to explain these observations, the resting form of the enzyme is inactive. Binding of BH4 stabilizes the inactive form, while binding of phenylalanine at a regulatory site activates the enzyme. The structure of the combined catalytic and regulatory domains of PheH provided a structural rationale for this model, by showing that residues 19-29 of the regulatory domain lie across the active site in the resting enzyme and presumably keep the enzyme from binding substrates [3]. Binding of phenylalanine at a regulatory site would then shift the enzyme to a conformation in which the active site was open. Direct structural evidence for this model is lacking. There is no reported structure of a form of PheH with both a regulatory domain and either phenylalanine or a pterin bound, so that the actual structural change that results in activation has not been established. In the absence of a direct structural support for a regulatory binding site for phenylalanine, the presence of such a site has come under question. A protein containing maltose binding protein fused to the N-terminus of the human PheH regulatory domain has been reported to bind radio-labeled phenylalanine [10]. In contrast, Thorolfsson et al. [11] could not detect any binding to the isolated regulatory domain by differential scanning calorimetry and concluded that activation is due to interactions between the active sites and that the role of the regulatory domain is to allow communication between active sites. Such a model would be consistent with the structure, which shows each regulatory domain interacting with two catalytic domains. To address the existence of a regulatory binding site for phenylalanine in PheH, we have expressed the isolated regulatory domain of rat PheH and analyzed the effects of phenylalanine on its structure. The results establish that there is a binding site for phenylalanine in this domain, consistent with the existence of an allosteric site for phenylalanine in PheH.