Thio Effects and an Unconventional Metal Ion Rescue in the Genomic Hepatitis Delta Virus Ribozyme

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• 作者：Pallavi Thaplyal ; Abir Ganguly ; Barbara L. Golden ; Sharon Hammes-Schiffer ; Philip C. Bevilacqua
• 刊名：Biochemistry
• 出版年：2013
• 出版时间：September 17, 2013
• 年：2013
• 卷：52
• 期：37
• 页码：6499-6514
• 全文大小：558K
• 年卷期：v.52,no.37(September 17, 2013)
• ISSN：1520-4995

文摘

Metal ion and nucleobase catalysis are important for ribozyme mechanism, but the extent to which they cooperate is unclear. A crystal structure of the hepatitis delta virus (HDV) ribozyme suggested that the pro-R_P oxygen at the scissile phosphate directly coordinates a catalytic Mg²⁺ ion and is within hydrogen bonding distance of the amine of the general acid C75. Prior studies of the genomic HDV ribozyme, however, showed neither a thio effect nor metal ion rescue using Mn²⁺. Here, we combine experiment and theory to explore phosphorothioate substitutions at the scissile phosphate. We report significant thio effects at the scissile phosphate and metal ion rescue with Cd²⁺. Reaction profiles with an S_P-phosphorothioate substitution are indistinguishable from those of the unmodified substrate in the presence of Mg²⁺ or Cd²⁺, supporting the idea that the pro-S_P oxygen does not coordinate metal ions. The R_P-phosphorothioate substitution, however, exhibits biphasic kinetics, with the fast-reacting phase displaying a thio effect of up to 5-fold and the slow-reacting phase displaying a thio effect of 1000-fold. Moreover, the fast- and slow-reacting phases give metal ion rescues in Cd²⁺ of up to 10- and 330-fold, respectively. The metal ion rescues are unconventional in that they arise from Cd²⁺ inhibiting the oxo substrate but not the R_P substrate. This metal ion rescue suggests a direct interaction of the catalytic metal ion with the pro-R_P oxygen, in line with experiments with the antigenomic HDV ribozyme. Experiments without divalent ions, with a double mutant that interferes with Mg²⁺ binding, or with C75 deleted suggest that the pro-R_P oxygen plays at most a redundant role in positioning C75. Quantum mechanical/molecular mechanical (QM/MM) studies indicate that the metal ion contributes to catalysis by interacting with both the pro-R_P oxygen and the nucleophilic 2鈥?hydroxyl, supporting the experimental findings.