Takusagawa F, Kamitori S, Markham G D
Department of Chemistry, University of Kansas, Lawrence, Kansas 66045-0046, USA.
Biochemistry. 1996 Feb 27;35(8):2586-96. doi: 10.1021/bi952604z.
S-Adenosylmethionine synthetase (MAT,ATP:L-methionine S-adenosltransferase, 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 (Pi) 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 MAT 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 Angstroms, c= 139.8 Angstroms, resolution limit 2.8 Angstroms; R O.19; Rfree 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 Pi by 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 Pi in 72 h under the same conditions as the crystallization of the enzyme. In the active site, the diphosphate moiety of ADP and Pi interacts extensively with 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.
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）在所有生物体中都起着核心代谢作用。MAT催化两步反应，该反应由ATP和L-甲硫氨酸合成S-腺苷甲硫氨酸（AdoMet）、焦磷酸（PPi）和正磷酸（Pi）。AdoMet是生物系统中的主要甲基供体。最近已确定了来自大肠杆菌的MAT的首个晶体结构[Takusagawa等人（1995年）《生物化学杂志》271，136 - 147]。为了阐明活性位点和可能的催化反应机制，已确定了在含有底物ATP（和溴代ATP）以及产物PPi的晶体中MAT的结构（空间群P6(2)22；晶胞a = b = 128.9埃，c = 139.8埃，分辨率极限2.8埃；R 0.19；Rfree 0.26）。该酶由四个相同的亚基组成；两个亚基形成一个球形二聚体，这些紧密结合的二聚体对形成一个四聚体酶。每个二聚体有两个位于亚基之间的活性位点。每个亚基由通过假三重对称性相互关联的三个结构域组成。晶体结构表明该酶将ATP水解为ADP和Pi。在活性位点发现了这些产物以及必需金属离子（K⁺和Mg²⁺）。这一相当意外的发现首先通过与PPi的复合物结构得到证实，随后通过高效液相色谱分析得到证实。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Pi。在活性位点，ADP和Pi的二磷酸部分与酶的两个亚基的氨基酸残基广泛相互作用，而腺嘌呤和核糖部分与酶的相互作用很小。结合ADP后酶的结构几乎没有变化。在已报道的来自多种生物体的14个MAT序列中，发现活性位点中涉及的所有氨基酸残基都是保守的。因此，本研究中确定的结构可作为MAT家族其他成员的模型。基于晶体结构，提出了AdoMet形成和三聚磷酸水解的催化反应机制。
