Identification of the Catalytic Mg2+ Ion in the Hepatitis Delta Virus Ribozyme

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• 作者：Ji Chen ; Abir Ganguly ; Zulaika Miswan ; Sharon Hammes-Schiffer ; Philip C. Bevilacqua ; Barbara L. Golden
• 刊名：Biochemistry
• 出版年：2013
• 出版时间：January 22, 2013
• 年：2013
• 卷：52
• 期：3
• 页码：557-567
• 全文大小：435K
• 年卷期：v.52,no.3(January 22, 2013)
• ISSN：1520-4995

文摘

The hepatitis delta virus ribozyme catalyzes an RNA cleavage reaction using a catalytic nucleobase and a divalent metal ion. The catalytic base, C75, serves as a general acid and has a pK_a shifted toward neutrality. Less is known about the role of metal ions in the mechanism. A recent crystal structure of the precleavage ribozyme identified a Mg²⁺ ion that interacts through its partial hydration sphere with the G25路U20 reverse wobble. In addition, this Mg²⁺ ion is in position to directly coordinate the nucleophile, the 2鈥?hydroxyl of U(鈭?), suggesting it can serve as a Lewis acid to facilitate deprotonation of the 2鈥?hydroxyl. To test the role of the active site Mg²⁺ ion, we replaced the G25路U20 reverse wobble with an isosteric A25路C20 reverse wobble. This change was found to significantly reduce the negative potential at the active site, as supported by electrostatics calculations, suggesting that active site Mg²⁺ binding could be adversely affected by the mutation. The kinetic analysis and molecular dynamics of the A25路C20 double mutant suggest that this variant stably folds into an active structure. However, pH鈥搑ate profiles of the double mutant in the presence of Mg²⁺ are inverted relative to the profiles for the wild-type ribozyme, suggesting that the A25路C20 double mutant has lost the active site metal ion. Overall, these studies support a model in which the partially hydrated Mg²⁺ positioned at the G25路U20 reverse wobble is catalytic and could serve as a Lewis acid, a Br酶nsted base, or both to facilitate deprotonation of the nucleophile.