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
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
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DOI：
10.1016/s0022-2836(02)00560-0
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发表时间：
2002-07-26
影响因子：
5.6
通讯作者：
Hough, E
中科院分区：
文献类型：
--
作者：
Andersen, OA;Flatmark, T;Hough, E
苯丙氨酸羟化酶催化 L-苯丙氨酸的立体特异性羟基化，这是该氨基酸降解的关键步骤。我们使用催化活性还原型辅因子解析了人苯丙氨酸羟化酶 (hPheOH) 截短形式 (DeltaN1-102/DeltaC428-452) 催化活性 Fe(II) 形式的三元复合物 (hPheOH-Fe(II).BH4.THA) 的晶体结构6(R)-L-erythro-5,6,7,8-四氢生物蝶呤 (BH4) 和 3-(2-噻吩基)-L-丙氨酸 (THA) 作为底物类似物。该类似物结合在催化铁原子的第二配位层中，噻吩环与 His285 的咪唑基团堆叠（平均晶面距离 3.8 埃），并具有氢键和疏水接触网络。该类似物与二元复合物 hPheOH-Fe(II)-BH 的结合引发了整个分子的结构变化，该分子采用了稍微更紧凑的结构。最大的变化发生在包含残基 131-155 的环区域，其中最大 r.m.s.位移（9.6 埃）位于 Tyr138。该环被重新折叠，使 Tyr138 18.5 埃的羟基氧原子更接近铁原子并进入活性位点。铁的几何形状是高度扭曲的方锥体，并且Glu330采用与hPheOH-Fe(II).BH4结构中观察到的构象不同的构象，具有双齿铁配位。 BH, 结合在催化铁原子的第二配位层中，相对于 hPheOH-Fe(II).BH, 结构，在 Glu286 和铁原子的方向上位移 2.6 埃，从而改变其氢键网络。三元复合物的活性位点结构为酶的底物特异性提供了新的见解，特别是对 L-酪氨酸的低亲和力。此外，该结构对催化机制和全长四聚酶被其底物激活的分子基础都有影响。 Tyr138 从表面位置移动到活性位点的构象变化可能反映了该残基可能的功能作用。 (C) 2002 Elsevier Science Ltd. 保留所有权利。
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 (DeltaN1-102/DeltaC428-452) 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 Angstrom) and with a network of hydrogen bonds and hydrophobic contacts. Binding of the analogue to the binary complex hPheOH-Fe(II)-BH, 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 Angstrom) is at Tyr138. This loop is refolded, bringing the hydroxyl oxygen atom of Tyr138 18.5 Angstrom 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. BH, binds in the second coordination sphere of the catalytic iron atom, and is displaced 2.6 Angstrom in the direction of Glu286 and the iron atom, relative to the hPheOH-Fe(II).BH, 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 implications both for the catalytic mechanism and the molecular basis the activation of the full-length tetrameric enzyme by its substrate. The conformational change, moving Tyr138 from a surface position into active site, may reflect a possible functional role for this residue. (C) 2002 Elsevier Science Ltd. All rights reserved.