Schalk-Hihi C, Markham G D
Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.
Biochemistry. 1999 Feb 23;38(8):2542-50. doi: 10.1021/bi9822933.
S-Adenosylmethionine (AdoMet) is the most widely used alkyl group donor in biological systems. The formation of AdoMet from ATP and L-methionine is catalyzed by S-adenosylmethionine synthetase (AdoMet synthetase). Elucidation of the conformations of enzyme-bound substrates, product, and inhibitors is important for the understanding of the catalytic mechanism of the enzyme and the design of new inhibitors. To obtain structural data for enzyme-bound substrates and product, we have used two-dimensional transferred nuclear Overhauser effect spectroscopy to determine the conformation of enzyme-bound AdoMet and 5'-adenylyl imidodiphosphate (AMPPNP). AMPPNP, an analogue of ATP, is resistant to the ATP hydrolysis activity of AdoMet synthetase because of the presence of a nonhydrolyzable NH-link between the beta- and gamma-phosphates but is a substrate for AdoMet formation during which tripolyphosphate is produced. AdoMet and AMPPNP both bind in an anti conformation about the glycosidic bond. The ribose rings are in C3'-exo and C4'-exo conformations in AdoMet and AMPPNP, respectively. The differences in ribose ring conformations presumably reflect the different steric requirements of the C5' substituents in AMPPNP and AdoMet. The NMR-determined conformations of AdoMet and AMPPNP were docked into the E. coli AdoMet synthetase active site taken from the enzyme.ADP. Pi crystal structure. Since there are no nonexchangeable protons either in the carboxy-terminal end of the methionine segment of AdoMet or in the tripolyphosphate segment of AMPPNP, these portions of the molecules were modeled into the enzyme active site. The interactions of AdoMet and AMPPNP with the enzyme predict the location of the methionine binding site and suggest how the positive charge formed on the sulfur during AdoMet synthesis is stabilized.
S-腺苷甲硫氨酸（AdoMet）是生物系统中使用最广泛的烷基供体。由ATP和L-甲硫氨酸形成AdoMet的反应由S-腺苷甲硫氨酸合成酶（AdoMet合成酶）催化。阐明酶结合底物、产物和抑制剂的构象对于理解酶的催化机制以及设计新的抑制剂很重要。为了获得酶结合底物和产物的结构数据，我们使用二维转移核Overhauser效应光谱来确定酶结合的AdoMet和5'-腺苷酰亚胺二磷酸（AMPPNP）的构象。AMPPNP是ATP的类似物，由于β-和γ-磷酸之间存在不可水解的NH键，它对AdoMet合成酶的ATP水解活性具有抗性，但它是AdoMet形成的底物，在此过程中会产生三聚磷酸。AdoMet和AMPPNP都以反式构象围绕糖苷键结合。核糖环在AdoMet中为C3'-外向构象，在AMPPNP中为C4'-外向构象。核糖环构象的差异可能反映了AMPPNP和AdoMet中C5'取代基不同的空间需求。通过核磁共振确定的AdoMet和AMPPNP的构象被对接至取自该酶.ADP.Pi晶体结构的大肠杆菌AdoMet合成酶活性位点。由于AdoMet的甲硫氨酸片段的羧基末端或AMPPNP的三聚磷酸片段中均不存在不可交换的质子，因此将分子的这些部分构建到酶活性位点中。AdoMet和AMPPNP与酶的相互作用预测了甲硫氨酸结合位点的位置，并表明了在AdoMet合成过程中硫上形成的正电荷是如何稳定的。
