Segal D M, Eichler D C
Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, Tampa 33612.
Arch Biochem Biophys. 1989 Dec;275(2):334-43. doi: 10.1016/0003-9861(89)90380-9.
The structural features of S-adenosyl-L-methionine (SAM)3 required for optimal binding to a nucleolar 2'-O-methyltransferase were elucidated using various analogs of SAM with modifications of the amino acid, sugar, sulfonium center, and base portions of the molecule. Equilibrium binding constants for SAM and each analog were determined by a nitrocellulose filter binding assay. To ensure the chiral and chemical purity of the 3H-labeled SAM used in the binding experiments, a cation-exchange HPLC procedure was developed to separate degradation products of SAM such as adenine and 5'-deoxy-5'-methylthioadenosine, as well as to separate the (S,S)-SAM from the biologically inactive (R,S)-SAM stereoisomer. Results from these studies demonstrated that S-adenosyl-L-homocysteine, a product of the methyltransferase reaction, bound equally as well as (S,S)-SAM, indicating that neither the charge nor the methyl group at the sulfonium center of (S,S)-SAM is essential for maximal binding. Other modifications of the sulfonium center demonstrated that a sulfur to carbon atom replacement had little effect on binding affinity, whereas substituting an ethyl group for the methyl group greatly reduced the binding affinity. In addition, the chirality at the sulfonium center was important. The naturally occurring S-chiral form had a 10-fold higher binding affinity than the R-chiral form. No significant stereospecificity was observed relative to the chiral alpha-carbon of the methionine moiety in SAM. The alpha-amino group of methionine and the 6-amino group of adenine were both required for maximal binding, while the loss of the 2'-hydroxyl group on the ribose moiety was not. Taken together, these results defined some of the specific geometric and functional group requirements which affect the specificity of interaction between S-adenosyl-L-methionine and the nucleolar 2'-O-methyltransferase.
利用S-腺苷-L-甲硫氨酸（SAM）的各种类似物，对其氨基酸、糖、锍中心和碱基部分进行修饰，阐明了SAM与核仁2'-O-甲基转移酶最佳结合所需的结构特征。通过硝酸纤维素滤膜结合试验测定SAM和每种类似物的平衡结合常数。为确保结合实验中使用的3H标记SAM的手性和化学纯度，开发了一种阳离子交换高效液相色谱法，用于分离SAM的降解产物，如腺嘌呤和5'-脱氧-5'-甲硫基腺苷，以及将（S,S）-SAM与无生物活性的（R,S）-SAM立体异构体分离。这些研究结果表明，甲基转移酶反应的产物S-腺苷-L-高半胱氨酸与（S,S）-SAM的结合能力相同，这表明（S,S）-SAM锍中心的电荷和甲基对于最大结合并非必不可少。锍中心的其他修饰表明，硫原子被碳原子取代对结合亲和力影响不大，而用乙基取代甲基则大大降低了结合亲和力。此外，锍中心的手性很重要。天然存在的S-手性形式的结合亲和力比R-手性形式高10倍。相对于SAM中甲硫氨酸部分的手性α-碳，未观察到明显的立体特异性。甲硫氨酸的α-氨基和腺嘌呤的6-氨基对于最大结合都是必需的，而核糖部分2'-羟基的缺失则不是必需的。综上所述，这些结果确定了一些特定的几何和官能团要求，这些要求影响S-腺苷-L-甲硫氨酸与核仁2'-O-甲基转移酶之间相互作用的特异性。
