Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-2063, United States.
Biochemistry. 2011 Nov 8;50(44):9424-33. doi: 10.1021/bi201157t. Epub 2011 Oct 17.
The hepatitis delta virus (HDV) ribozyme and related RNAs are widely dispersed in nature. This RNA is a small nucleolytic ribozyme that self-cleaves to generate products with a 2',3'-cyclic phosphate and a free 5'-hydroxyl. Although small ribozymes are dependent on divalent metal ions under biologically relevant buffer conditions, they function in the absence of divalent metal ions at high ionic strengths. This characteristic suggests that a functional group within the covalent structure of small ribozymes is facilitating catalysis. Structural and mechanistic analyses have demonstrated that the HDV ribozyme active site contains a cytosine with a perturbed pK(a) that serves as a general acid to protonate the leaving group. The reaction of the HDV ribozyme in monovalent cations alone never approaches the velocity of the Mg(2+)-dependent reaction, and there is significant biochemical evidence that a Mg(2+) ion participates directly in catalysis. A recent crystal structure of the HDV ribozyme revealed that there is a metal binding pocket in the HDV ribozyme active site. Modeling of the cleavage site into the structure suggested that this metal ion can interact directly with the scissile phosphate and the nucleophile. In this manner, the Mg(2+) ion can serve as a Lewis acid, facilitating deprotonation of the nucleophile and stabilizing the conformation of the cleavage site for in-line attack of the nucleophile at the scissile phosphate. This catalytic strategy had previously been observed only in much larger ribozymes. Thus, in contrast to most large and small ribozymes, the HDV ribozyme uses two distinct catalytic strategies in its cleavage reaction.
乙型肝炎 Delta 病毒 (HDV) 核酶和相关 RNA 广泛存在于自然界中。这种 RNA 是一种小分子核酸酶核酶，它通过自我切割产生具有 2'、3'-环磷酸和游离 5'-羟基的产物。尽管小分子核酶在生物学相关缓冲条件下依赖于二价金属离子，但它们在高离子强度下没有二价金属离子也能发挥作用。这一特性表明，小分子核酶共价结构内的一个功能基团促进了催化作用。结构和机制分析表明，HDV 核酶活性位点包含一个 pKa 受到干扰的胞嘧啶，它作为一种广义酸，将离去基团质子化。在单价阳离子中进行的 HDV 核酶反应从未接近依赖 Mg2+的反应速度，并且有大量生化证据表明 Mg2+离子直接参与催化。HDV 核酶的最近晶体结构揭示了 HDV 核酶活性位点内存在一个金属结合口袋。将切割位点建模到结构中表明，这种金属离子可以直接与可切割的磷酸酯和亲核试剂相互作用。通过这种方式，Mg2+离子可以作为路易斯酸，促进亲核试剂的去质子化，并稳定切割位点的构象，使亲核试剂在线攻击可切割的磷酸酯。这种催化策略以前只在更大的核酶中观察到。因此，与大多数大型和小型核酶不同，HDV 核酶在其切割反应中使用两种不同的催化策略。
