Abstract
Phenylalanine hydroxylase (PAH) catalyzes the conversion of L-Phe to L-Tyr. Defects in PAH activity, caused by mutations in the human gene, result in the autosomal recessively inherited disease hyperphenylalaninemia. PAH activity is regulated by multiple factors, including phosphorylation and ligand binding. In particular, PAH displays positive cooperativity for L-Phe, which is proposed to bind the enzyme on an allosteric site in the N-terminal regulatory domain (RD), also classified as an ACT domain. This domain is found in several proteins and is able to bind amino acids. We used molecular dynamics simulations to obtain dynamical and structural insights into the isolated RD of PAH. Here we show that the principal motions involve conformational changes leading from an initial open to a final closed domain structure. The global intrinsic motions of the RD are correlated with exposure to solvent of a hydrophobic surface, which corresponds to the ligand binding-site of the ACT domain. Our results strongly suggest a relationship between the Phe-binding function and the overall dynamic behaviour of the enzyme. This relationship may be affected by structure-disturbing mutations. To elucidate the functional implications of the mutations, we investigated the structural effects on the dynamics of the human RD PAH induced by six missense hyperphenylalaninemia-causing mutations, namely p.G46S, p.F39C, p.F39L, p.I65S, p.I65T and p.I65V. These studies showed that the alterations in RD hydrophobic interactions induced by missense mutations could affect the functionality of the whole enzyme.
Highlights
Phenylalanine hydroxylase (PAH) is an iron-containing enzyme, mainly expressed in liver, that catalyzes the conversion of the essential amino acid L-Phe into LTyr utilizing the cofactor 6R-L-erythro-tetrahydrobiopterin (BH4) and dioxygen [1,2]
Defects in PAH enzymatic activity caused by mutations in the human gene result in an autosomal recessively inherited disorder of amino acid metabolism known as hyperphenylalaninemia (HPA)
We investigated the structural and dynamic effects induced by six selected missense disease-causing mutations found in young PKU patients (p.G46S, p.F39C, p.F39L, p.I65S, p.I65T and p.I65V) [28] and located on the regulatory domain (RD) of the human PAH (hPAH) enzyme
Summary
Phenylalanine hydroxylase (PAH) is an iron-containing enzyme, mainly expressed in liver, that catalyzes the conversion of the essential amino acid L-Phe (hereafter referred to as ‘‘Phe’’) into LTyr utilizing the cofactor 6R-L-erythro-tetrahydrobiopterin (BH4) and dioxygen [1,2]. Defects in PAH enzymatic activity caused by mutations in the human gene result in an autosomal recessively inherited disorder of amino acid metabolism known as hyperphenylalaninemia (HPA). Impaired PAH function results in the accumulation of high levels of blood plasma phenylalanine and of its neurotoxic metabolites [3]. Mutations in the human PAH gene lead to a variety of clinical and biochemical phenotypes that differ in severity [4,5]: mild hyperphenylalaninemia, mild phenylketonuria and classical phenylketonuria (PKU). Diagnosis and prompt intervention has allowed most individuals with PKU to avoid severe mental disability [6]. To prevent mental retardation due to the buildup of neurotoxic metabolites of Phe, patients with severe PKU must be treated with a low-Phe diet starting early in life [7]. Phe excess is a main cause of such alterations in brain function as spatial learning deficits and long-term potentiation [8], indirectly indicating that a single amino acid, such as Phe, can alter physiological homeostasis probably by unfavorably interacting with the functionality of cell proteins
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