The specificity of interaction between S-adenosyl-L-methionine and a nucleolar 2'-O-methyltransferase.
The specificity of interaction between S-adenosyl-L-methionine and a nucleolar 2'-O-methyltransferase.
复制标题
S-腺苷-L-甲硫氨酸和核仁 2-O-甲基转移酶之间相互作用的特异性。
DOI：
10.1016/0003-9861(89)90380-9
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发表时间：
1989
影响因子：
3.9
通讯作者：
Eichler,DC
中科院分区：
文献类型：
--
作者：
Segal,DM;Eichler,DC
利用不同的S-腺苷-L-甲硫氨酸（SAM）类似物，通过对氨基酸、糖、锍中心和碱基部分的修饰，阐明了SAM 3与核仁2′-O-甲基转移酶最佳结合所需的结构特征。SAM和每种类似物的平衡结合常数通过硝酸纤维素滤膜结合测定法测定。为了保证3 H标记SAM的手性和化学纯度，我们建立了阳离子交换高效液相色谱分离SAM降解产物腺嘌呤和5′-脱氧-5 ′-甲硫基腺苷的方法，并将（S，S）-SAM与无生物活性的（R，S）-SAM立体异构体分离。从这些研究的结果表明，S-腺苷-L-高半胱氨酸，甲基转移酶反应的产物，结合同样以及（S，S）-SAM，表明无论是电荷或甲基在锍中心的（S，S）-SAM是必不可少的最大结合。锍中心的其他修饰表明，硫到碳原子的取代对结合亲和力几乎没有影响，而用乙基取代甲基则大大降低了结合亲和力。此外，在锍中心的手性是重要的。天然存在的S-手性形式具有比R-手性形式高10倍的结合亲和力。相对于SAM中甲硫氨酸部分的手性α-碳，未观察到显著的立体特异性。甲硫氨酸的α-氨基和腺嘌呤的6-氨基都是最大结合所必需的，而核糖部分上2′-羟基的损失则不是。总之，这些结果定义了一些特定的几何和官能团的要求，影响S-腺苷-L-蛋氨酸和核仁2′-O-甲基转移酶之间的相互作用的特异性。
The structural features ofS-adenosyl-l-methionine (SAM)3required 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 the3H-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 thatS-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 occurringS-chiral form had a 10-fold higher binding affinity than theR-chiral form. No significant stereospecificity was observed relative to the chiral α-carbon of the methionine moiety in SAM. The α-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 betweenS-adenosyl-l-methionine and the nucleolar 2′-O-methyltransferase.