Crystal Structure of the Ternary Complex of the Catalytic Domain of Human Phenylalanine Hydroxylase with Tetrahydrobiopterin and 3-(2-Thienyl)-l-alanine, and its Implications for the Mechanism of Catalysis and Substrate Activation

Abstract

Phenylalanine hydroxylase catalyzes the stereospecific hydroxylation of l-phenylalanine, the committed step in the degradation of this amino acid. We have solved the crystal structure of the ternary complex (hPheOH–Fe(II)·BH₄·THA) of the catalytically active Fe(II) form of a truncated form (ΔN1–102/ΔC428–452) of human phenylalanine hydroxylase (hPheOH), using the catalytically active reduced cofactor 6(R)-l-erythro-5,6,7,8-tetrahydrobiopterin (BH₄) and 3-(2-thienyl)-l-alanine (THA) as a substrate analogue. The analogue is bound in the second coordination sphere of the catalytic iron atom with the thiophene ring stacking against the imidazole group of His285 (average interplanar distance 3.8 Å) and with a network of hydrogen bonds and hydrophobic contacts. Binding of the analogue to the binary complex hPheOH–Fe(II)·BH₄ triggers structural changes throughout the entire molecule, which adopts a slightly more compact structure. The largest change occurs in the loop region comprising residues 131–155, where the maximum r.m.s. displacement (9.6 Å) is at Tyr138. This loop is refolded, bringing the hydroxyl oxygen atom of Tyr138 18.5 Å closer to the iron atom and into the active site. The iron geometry is highly distorted square pyramidal, and Glu330 adopts a conformation different from that observed in the hPheOH–Fe(II)·BH₄ structure, with bidentate iron coordination. BH₄ binds in the second coordination sphere of the catalytic iron atom, and is displaced 2.6 Å in the direction of Glu286 and the iron atom, relative to the hPheOH–Fe(II)·BH₄ structure, thus changing its hydrogen bonding network. The active-site structure of the ternary complex gives new insight into the substrate specificity of the enzyme, notably the low affinity for l-tyrosine. Furthermore, the structure has implications both for the catalytic mechanism and the molecular basis for the activation of the full-length tetrameric enzyme by its substrate. The large conformational change, moving Tyr138 from a surface position into the active site, may reflect a possible functional role for this residue.

Introduction

The non-heme iron enzyme phenylalanine hydroxylase (PheOH, phenylalanine 4-monooxygenase, EC 1.14.16.1) catalyzes the hydroxylation of the essential aromatic amino acid l-phenylalanine (l-Phe) to l-tyrosine (l-Tyr) in the presence of the specific pterin cofactor 6(R)-l-erythro-5,6,7,8-tetrahydrobiopterin (BH₄) and dioxygen. The reaction is the rate-limiting step in the degradation of l-Phe to carbon dioxide and water.¹ Inborn errors that reduce or destroy the activity of the enzyme are responsible for the human autosomal recessive disease phenylketonuria (PKU)/hyperphenylalaninemia (HPA). The disease causes elevated concentrations of l-Phe in the blood, which can impair the normal development of the brain and cause severe mental retardation. In most of Europe, approximately 1 in 10,000 live births reportedly has the disorder² and more than 400 different mutations are associated with PKU/HPA.³† Most of the mutations are found in the catalytic domain³ and they demonstrate different clinical, metabolic and enzymatic phenotypes.4., 5. Recent crystallographic studies on human phenylalanine hydroxylase (hPheOH)6., 7., 8., 9. and rat phenylalanine hydroxylase (rPheOH)¹⁰ have made it possible to define the structural phenotypes of the different genotypes.¹¹ A limitation in the assignment of the structural phenotypes has been that they have been based on the structures of catalytically inactive Fe(III) forms of the enzyme, which also lack structural information on substrate binding. Following our recently solved crystal structure of the catalytically active Fe(II) form of the truncated form ΔN1–102/ΔC428–452-hPheOH and its binary complex with the reduced pterin cofactor (BH₄),¹² we now present the crystal structure of a ternary complex with the substrate analogue 3-(2-thienyl)-l-alanine (THA). THA is a substrate for rPheOH¹³ and hPheOH¹⁴ and binds competitively to l-Phe at the active site.¹⁵ Binding of the substrate analogue also triggers a conformational change similar to that observed upon binding of l-Phe.14., 16., 17.

The structure reveals the binding sites of the pterin cofactor and the substrate under near-turnover conditions, i.e. in the absence of dioxygen, and provides new insights into the substrate specificity and catalytic mechanism of the enzyme. It shows that substrate binding triggers a substantial structural change in the catalytic domain, particularly in the active-site region. This change may represent the “epicenter” of the global conformational transition and catalytic activation that occurs in the full-length tetrameric enzyme upon substrate binding.¹⁸