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首页> 外文期刊>Journal of Molecular Biology >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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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.

机译：人苯丙氨酸羟化酶与四氢生物蝶呤和3-（2-噻吩基）-L-丙氨酸催化域的三元配合物的晶体结构，及其对催化和底物活化机理的启示。

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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).BH(4).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 (BH(4)) 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 3.8A) and with a network of hydrogen bonds and hydrophobic contacts. Binding of the analogue to the binary complex hPheOH-Fe(II).BH(4) 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 (9.6A) is at Tyr138. This loop is refolded, bringing the hydroxyl oxygen atom of Tyr138 18.5A 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).BH(4) structure, with bidentate iron coordination. BH(4) binds in the second coordination sphere of the catalytic iron atom, and is displaced 2.6A in the direction of Glu286 and the iron atom, relative to the hPheOH-Fe(II).BH(4) 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.
机译：苯丙氨酸羟化酶催化L-苯丙氨酸的立体定向羟基化，这是该氨基酸降解的重要步骤。我们已经解决了人苯丙氨酸的截短形式（DeltaN1-102 / DeltaC428-452）的催化活性Fe（II）形式的三元配合物（hPheOH-Fe（II）.BH（4）.THA）的晶体结构羟化酶（hPheOH），使用催化活性降低的辅因子6（R）-L-赤型5,6,7,8-四氢生物蝶呤（BH（4））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个配位域中，噻吩环堆积在His285的咪唑基上（平均晶面间距为3.8A），并带有氢键和疏水接触网络。该类似物与二元复合物hPheOH-Fe（II）.BH（4）的结合会触发整个分子的结构变化，从而采用稍微更紧凑的结构。最大变化发生在包含残基131-155的环区域中，其中最大r.m.s.排量（9.6A）在Tyr138。重新折叠此环，使Tyr138 18.5A的羟基氧原子更靠近铁原子并进入活性位点。铁的几何形状是高度扭曲的方形锥体，Glu330的构象与hPheOH-Fe（II）.BH（4）结构中观察到的构象不同，具有二齿铁配位。 BH（4）结合在催化铁原子的第二个配位域中，并相对于hPheOH-Fe（II）.BH（4）结构在Glu286和铁原子的方向上位移2.6A。氢键网络。三元复合物的活性位点结构使人们对酶的底物特异性有了新的认识，特别是对L-酪氨酸的亲和力低。此外，该结构对于其底物激活全长四聚酶的催化机理和分子基础都有影响。大的构象变化将Tyr138从表面位置移动到活性位点，可能反映了此残基的可能功能。

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