Structure and function of S-adenosylmethionine synthetase: Crystal structures of S-adenosylmethionine synthetase with ADP, BrADP, and PPi at 2.8 angstrom resolution
Structure and function of S-adenosylmethionine synthetase: Crystal structures of S-adenosylmethionine synthetase with ADP, BrADP, and PPi at 2.8 angstrom resolution
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DOI：
10.1021/bi952604z
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发表时间：
1996-02-27
期刊：
影响因子：
2.9
通讯作者：
Markham, GD
中科院分区：
文献类型：
--
作者：
Takusagawa, F;Kamitori, S;Markham, GD
S-腺苷甲硫氨酸合成酶（MAT，ATP：L-甲硫氨酸S-腺苷转移酶，EC 2.5.1.6）在所有生物体中起着重要的代谢作用。MAT催化从ATP和L-甲硫氨酸合成S-腺苷甲硫氨酸（ATP Met）、焦磷酸盐（PPi）和正磷酸盐（P-i）的两步反应。蛋氨酸是生物系统中主要的甲基供体。最近已经确定了来自大肠杆菌的MAT的第一种晶体结构[Takusagawa等人（1995）J.Biol.Chem.271，136-147]。为了阐明活性中心和可能的催化反应机理，测定了以底物ATP（和BrATP）和产物PPi生长的晶体中的M. 4 T结构（空间群P6（2）22;晶胞a = B = 128.9埃，c = 139.8埃，分辨极限2.8埃; R 0.19; R（free）0.26）。该酶由四个相同的亚基组成;两个亚基形成球形二聚体，这些紧密结合的二聚体成对形成四聚体酶。每个二聚体具有两个位于亚基之间的活性位点，每个亚基由三个通过假三重对称性彼此相关的结构域组成。晶体结构表明，ATP分子在酶的作用下水解为ADP和P-i b4，这些产物与必需的金属离子（K+和Mg ~（2+））一起沿着于活性中心。这一相当出乎意料的发现首先通过与PPi的复合物的结构证实，随后通过HPLC分析证实。在与酶结晶相同的条件下，该酶在72小时内将ATP水解为ADP和P-i。在活性位点中，ADP和Pi的二磷酸部分与来自酶的两个亚基的氨基酸残基广泛地相互作用，而腺嘌呤和核糖部分与酶几乎没有相互作用。结合ADP后酶的结构变化不大。所有参与活性位点的氨基酸残基在广泛的生物体的MAT的14个已报道的序列中被发现是保守的。因此，本研究中确定的结构可以用作MAT家族其他成员的模型。根据晶体结构，提出了三聚磷酸盐形成和水解的催化反应机理。
S-Adenosylmethionine synthetase (MAT, ATP:L-methionine S-adenosyltransferase, EC 2.5.1.6) plays a central metabolic role in all organisms. MAT catalyzes the two-step reaction which synthesizes S-adenosylmethionine (AdoMet), pyrophosphate (PPi), and orthophosphate (P-i) from ATP and L-methionine. AdoMet is the primary methyl group donor in biological systems. The first crystal structure of MAT from Escherichia coli has recently been determined [Takusagawa et al. (1995) J. Biol. Chem. 271, 136-147]. In order to elucidate the active site and possible catalytic reaction mechanism, the M.4T structures in the crystals grown with the substrate ATP (and BrATP) and the product PPi have been determined (space group P6(2)22; unit cell a = b = 128.9 Angstrom, c = 139.8 Angstrom, resolution limit 2.8 Angstrom; R 0.19; R(free) 0.26). The enzyme consists of four identical subunits; two subunits form a spherical dimer, and pairs of these tightly bound dimers form a tetrameric enzyme. Each dimer has two active sites which are located between the subunits, Each subunit consists of three domains related to each other by a pseudo 3-fold symmetry. The crystal structures showed that the ATP molecules were hydrolyzed to ADP and P-i b4 the enzyme, Those products were found at the active site along with the essential metal ions (K+ and Mg2+). This rather unexpected finding was first confirmed by the structure of the complex with PPi and later by an HPLC analysis, The enzyme hydrolyzed ATP to ADP and P-i in 72 h under the same conditions as the crystallization of the enzyme. In the active site, the diphosphate moiety of ADP and P-i interacts extensively with the amino acid residues from the two subunits of the enzyme, whereas the adenine and ribose moieties have little interaction with the enzyme. The enzyme structure is little changed upon binding ADP. All amino acid residues involved in the active site are found to be conserved in the 14 reported sequences of MAT from a wide range of organisms. Thus the structure determined in this study can be utilized as a model for other members of the MAT family. On the basis of the crystal structures, the catalytic reaction mechanisms of AdoMet formation and hydrolysis of tripolyphosphate are proposed.