Mechanism of recognition of ribosomal substrate by site-specific RNA N-glycosidases

蓖麻毒素A链是一种RNA N-糖苷酶，它通过去除28S rRNA中高度保守的腺苷来灭活核糖体。化学修饰研究表明，位于活性部位裂隙外的某些精氨酸残基也参与了酶的活性。为了证实这一建议，进行了定点突变。野生型和突变型蛋白均在大肠杆菌中表达，纯化后进行了酶活性检测。Arg213向Ser的转化使kcat降低了15倍，而对Km没有影响，这表明Arg213具有催化作用。Arg196转换为Gln的活性降低了330倍，而Arg235转换为Ser的单独转换导致了更严重的活性损失(17000倍)。突变蛋白的圆二色谱和荧光分析表明，Trp211的二级结构和裂隙底部的环境都不受每个突变的影响。Arg213的侧链与裂隙边缘相邻。Arg196和Arg235位于裂隙的另一侧。这些结果支持活性部位外的Arg残基也参与了酶的作用，为了解蓖麻毒素A链的酶作用机制提供了有价值的信息。

Ricin A-chain is an RNA N-glycosidase that inactivates ribosomes by depurination of a highly conserved adenosine in 28S rRNA.Chemical modification studies suggested that certain arginine residues, located outside the active site cleft, are also involved in the enzymatic activity. To confirm this suggestion, site-directed mutagenesis was performed. Both the wild-type and mutant proteins were expressed in Escherichia coli, purified and tested for enzymatic activity. Conversion of Arg213 to Ser reduced kcat 15-fold without effect on Km, suggesting that Arg213 plays a role in catalysis. Conversion of Arg196 to Gln reduced the activity 330-fold, and the individual conversion of Arg235 to Ser caused a more drastic loss (17000-fold) in activity. Circular dichroism and fluorescence analyzes of the mutant proteins suggested that neither the secondary structure nor the environment of Trp211 at the bottom of the cleft was affected upon each mutation. The side chain of Arg213 is adjacent to the edge of the cleft. Arg196 and Arg235 are located on the other side of the cleft. These results support the idea that the Arg residues outside the active site cleft are also involved in the enzymatic action, providing valuable information to understand the mechanism of enzymatic action of ricin A-chain.