González B, Pajares M A, Hermoso J A, Alvarez L, Garrido F, Sufrin J R, Sanz-Aparicio J
Grupo de Cristalografía Macromolecular y Biología Estructural, Instituto de Química-Física Rocasolano CSIC, Serrano 119, 28006 Madrid, Spain.
J Mol Biol. 2000 Jul 7;300(2):363-75. doi: 10.1006/jmbi.2000.3858.
Most of the transmethylation reactions use the same methyl donor, S-adenosylmethionine (SAM), that is synthesised from methionine and ATP by methionine adenosyltransferase (MAT). In mammals, two MAT enzymes have been detected, one ubiquitous and another liver specific. The liver enzyme exists in two oligomeric forms, a tetramer (MAT I) and a dimer (MAT III), MAT I being the one that shows a higher level of affinity for methionine but a lower SAM synthesis capacity. We have solved the crystal structure of rat liver MAT I at 2.7 A resolution, complexed with a methionine analogue: l-2-amino-4-methoxy-cis-but-3-enoic acid (l-cisAMB). The enzyme consists of four identical subunits arranged in two tight dimers that are related by crystallographic 2-fold symmetry. The crystal structure shows the positions of the relevant cysteine residues in the chain, and that Cys35 and Cys61 are perfectly oriented for forming a disulphide link. This result leads us to propose a hypothesis to explain the control of MAT I/III exchange and hence, the effects observed on activity. We have identified the methionine-binding site into the active-site cavity, for the first time. The l-cisAMB inhibitor is stacked against Phe251 aromatic ring in a rather planar conformation, and its carboxylate group coordinates a Mg(2+), which, in turn, is linked to Asp180. The essential role of the involved residues in MAT activity has been confirmed by site-directed mutagenesis. Phe251 is exposed to solvent and is located in the beginning of the flexible loop Phe251-Ala260 that is connecting the N-terminal domain to the central domain. We postulate that a conformational change may take place during the enzymatic reaction and this is possibly the reason of the unusual two-step mechanism involving tripolyphosphate hydrolysis. Other important mechanistic implications are discussed on the light of the results. Moreover, the critical role that certain residues identified in this study may have in methionine recognition opens further possibilities for rational drug design.
大多数转甲基反应使用相同的甲基供体，即由蛋氨酸和ATP通过蛋氨酸腺苷转移酶（MAT）合成的S-腺苷甲硫氨酸（SAM）。在哺乳动物中，已检测到两种MAT酶，一种普遍存在，另一种为肝脏特异性。肝脏酶以两种寡聚形式存在，一种四聚体（MAT I）和一种二聚体（MAT III），MAT I对蛋氨酸具有较高的亲和力，但SAM合成能力较低。我们已经解析了大鼠肝脏MAT I与蛋氨酸类似物：l-2-氨基-4-甲氧基-顺式-丁-3-烯酸（l-cisAMB）复合后的2.7埃分辨率晶体结构。该酶由四个相同的亚基组成，排列成两个紧密的二聚体，通过晶体学2次对称性相关联。晶体结构显示了链中相关半胱氨酸残基的位置，并且Cys35和Cys61的取向完美地适于形成二硫键。这一结果使我们提出一个假说来解释MAT I/III交换的控制，从而解释观察到的对活性的影响。我们首次在活性位点腔内鉴定出蛋氨酸结合位点。l-cisAMB抑制剂以相当平面的构象堆积在Phe251芳香环上，其羧酸盐基团与Mg(2+)配位，而Mg(2+)又与Asp180相连。通过定点诱变已证实了所涉及残基在MAT活性中的重要作用。Phe251暴露于溶剂中，位于连接N端结构域和中央结构域的柔性环Phe251-Ala260的起始处。我们推测在酶促反应过程中可能发生构象变化，这可能是涉及三聚磷酸水解的异常两步机制的原因。根据结果讨论了其他重要的机制意义。此外，本研究中鉴定出的某些残基在蛋氨酸识别中可能具有的关键作用为合理药物设计开辟了进一步的可能性。
