Roday Setu, Saen-oon Suwipa, Schramm Vern L
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
Biochemistry. 2007 May 29;46(21):6169-82. doi: 10.1021/bi0621821. Epub 2007 May 4.
8-Vinyl-2'-deoxyadenosine (8vdA) is a fluorophore with a quantum yield comparable to that of 2-aminopurine nucleoside. 8vdA was incorporated into a 10-mer stem-tetraloop RNA (8vdA-10) structure for characterization of the properties of the base, 8-vinyladenine (8-vA), with respect to adenine as a substrate or inhibitor for ribosome-inactivating proteins. Ricin toxin A-chain (RTA) and pokeweed antiviral protein (PAP) catalyze the release of adenine from a specific adenosine on a stem-tetraloop (GAGA) sequence at the elongation factor (eEF2) binding site of the 28S subunit of eukaryotic ribosomes, thereby arresting translation. RTA does not catalyze the release of 8-vinyladenine from 8vdA-10. Molecular dynamics simulations implicate a role for Arg180 in oxacarbenium ion destabilization and the lack of catalysis. However, 8vdA-10 is an active site analogue and inhibits RTA with a Ki value of 2.4 microM. Adenine is also released from the second adenosine in the modified tetraloop, demonstrating an alternative mode for the binding of this motif in the RTA active site. The 8vdA analogue defines the specificities of RTA for the two adenylate depurination sites in a RNA substrate with a GAGA tetraloop. The rate of nonenzymatic acid-catalyzed solvolysis of 8-vinyladenine from the stem-loop RNA is described. Unlike RTA, PAP catalyzes the slow release of 8-vinyladenine from 8vdA-10. The isolation of 8-vA and its physicochemical characterization is described.
8-乙烯基-2'-脱氧腺苷（8vdA）是一种荧光团，其量子产率与2-氨基嘌呤核苷相当。8vdA被并入一个10聚体茎-四环RNA（8vdA-10）结构中，以表征碱基8-乙烯基腺嘌呤（8-vA）作为核糖体失活蛋白的底物或抑制剂相对于腺嘌呤的性质。蓖麻毒素A链（RTA）和商陆抗病毒蛋白（PAP）催化腺嘌呤从真核核糖体28S亚基延伸因子（eEF2）结合位点的茎-四环（GAGA）序列上的特定腺苷释放，从而阻止翻译。RTA不催化8vdA-10中8-乙烯基腺嘌呤的释放。分子动力学模拟表明Arg180在氧碳鎓离子去稳定化和缺乏催化作用中起作用。然而，8vdA-10是一种活性位点类似物，以2.4 microM的Ki值抑制RTA。腺嘌呤也从修饰四环中的第二个腺苷释放，证明了该基序在RTA活性位点结合的另一种模式。8vdA类似物定义了RTA对具有GAGA四环的RNA底物中两个腺苷酸脱嘌呤位点的特异性。描述了从茎环RNA中非酶促酸催化的8-乙烯基腺嘌呤溶剂解速率。与RTA不同，PAP催化8vdA-10中8-乙烯基腺嘌呤的缓慢释放。描述了8-vA的分离及其物理化学表征。
