2.0 Ã… resolution crystal structures of the ternary complexes of human phenylalanine hydroxylase catalytic domain with tetrahydrobiopterin and 3-(2-thienyl)-L-alanine or L-norleucine:: Substrate specificity and molecular motions related to substrate binding
2.0 Ã… resolution crystal structures of the ternary complexes of human phenylalanine hydroxylase catalytic domain with tetrahydrobiopterin and 3-(2-thienyl)-L-alanine or L-norleucine:: Substrate specificity and molecular motions related to substrate binding
复制标题
DOI：
10.1016/j.jmb.2003.09.004
复制
发表时间：
2003-10-31
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;Stokka, AJ;Hough, E
在2.0埃分辨率下测定了人苯丙氨酸羟化酶（hPheOH）的催化结构域与生理辅因子6（R）-L-β-5，6，7，8-四氢生物蝶呤（BH_4）和底物类似物3-（2-噻吩基）-L-丙氨酸（THA）或L-正亮氨酸（NLE）复合物的晶体结构。三元THA复合物证实了先前的2.5埃结构，并且三元NLE复合物显示在NLE结合时发生与THA所观察到的那些类似的大的构象变化。这两种结构都表明，底物结合引发整个原聚体的结构变化，包括Tyr 138从表面位置到活性位点的掩埋位置的位移，其羟基的最大位移为20.7埃。两个铰链弯曲区域，集中在Leu 197和Asn 223，在基板结合后，产生进一步的大的结构变化的C末端的部分。因此，THA/L-Phe结合到活性位点可能代表在全长四聚体酶中观察到的全局构象变化的震中。THA和NLE的羧基和氨基在两种结构中的位置相同，支持这些基团在底物结合中具有关键重要性的结论，从而解释了在人工活化形式的酶中观察到的广泛的非生理底物特异性。然而，以NLE为底物的比活性仅为THA的5%左右，这是由不同的结合亲和力和不同的催化周转率解释的。(C)2003 Elsevier Ltd.保留所有权利。
The crystal structures of the catalytic domain of human phenylalanine hydroxylase (hPheOH) in complex with the physiological cofactor 6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) and the substrate analogues 3-(2-thienyl)-L-alanine (THA) or L-norleucine (NLE) have been determined at 2.0 Angstrom resolution. The ternary THA complex confirms a previous 2.5 Angstrom structure, and the ternary NLE complex shows that similar large conformational changes occur on binding of NLE as those observed for THA. Both structures demonstrate that substrate binding triggers structural changes throughout the entire protomer, including the displacement of Tyr138 from a surface position to a buried position at the active site, with a maximum displacement of 20.7 Angstrom for its hydroxyl group. Two hinge-bending regions, centred at Leu197 and Asn223, act in consort upon substrate binding to create further large structural changes for parts of the C terminus. Thus, THA/L-Phe binding to the active site is likely to represent the epicentre of the global conformational changes observed in the full-length tetrameric enzyme. The carboxyl and amino groups of THA and NLE are positioned identically in the two structures, supporting the conclusion that these groups are of key importance in substrate binding, thus explaining the broad non-physiological substrate specificity observed for artificially activated forms of the enzyme. However, the specific activity with NLE as the substrate was only about 5% of that with THA, which is explained by the different affinities of binding and different catalytic turnover. (C) 2003 Elsevier Ltd. All rights reserved.