Amukele Tim K, Roday Setu, Schramm Vern L
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
Biochemistry. 2005 Mar 22;44(11):4416-25. doi: 10.1021/bi0474362.
Ricin toxin A-chain (RTA) depurinates a single adenylate on a GAGA stem-loop region of eukaryotic 28S RNA, making it a potent toxin. Steady state rate analysis is used to establish the kinetic parameters for depurination of short RNA, DNA, and RNA-DNA hybrids of GAGA linear segments and stem-loop regions as substrates for RTA. Both stem and tetraloop structures are essential for action on RNA. For DNA stem-loop substrates, stem stability plays a small role in enhancing catalytic turnover but can enhance binding by up to 3 orders of magnitude. DNA sequences of d[GAGA] without stem-loop structures are found to be slow substrates for RTA. In contrast, equivalent RNA sequences exhibit no activity with RTA. Introduction of a deoxyadenosine at the depurination site of short RNA oligonucleotides restores catalytic function. NMR analysis indicates that the short, nonsubstrate GAGA is converted to substrate in GdAGA by the presence of a more flexible ribosyl group at the deoxyadenosine site. Conversion between C2'-endo and C2'-exo conformations at the deoxyadenosine site moves the 3'- and 5'-phosphorus atoms by 1.1 A, and the former is proposed to place them in a catalytically favorable configuration. The ability to use short RNA-DNA hybrids as substrates for RTA permits exploration of related structures to function as substrates and inhibitors.
蓖麻毒素A链（RTA）可使真核生物28S RNA的GAGA茎环区域上的单个腺苷酸脱嘌呤，使其成为一种强效毒素。稳态速率分析用于确定以GAGA线性片段和茎环区域的短RNA、DNA及RNA-DNA杂交体作为RTA底物进行脱嘌呤反应的动力学参数。茎结构和四环结构对于RTA作用于RNA都是必不可少的。对于DNA茎环底物，茎的稳定性在增强催化周转方面作用较小，但可使结合增强多达3个数量级。发现没有茎环结构的d[GAGA] DNA序列是RTA的低效底物。相比之下，等效的RNA序列对RTA无活性。在短RNA寡核苷酸的脱嘌呤位点引入脱氧腺苷可恢复催化功能。核磁共振分析表明，短的非底物GAGA通过脱氧腺苷位点处更灵活的核糖基团的存在而在GdAGA中转化为底物。脱氧腺苷位点处C2'-内型和C2'-外型构象之间的转换使3'-和5'-磷原子移动了1.1埃，并且有人提出前者将它们置于催化有利的构型中。将短RNA-DNA杂交体用作RTA底物的能力允许探索相关结构作为底物和抑制剂的功能。
