4.4 Article

Evidence That Nucleophile Deprotonation Exceeds Bond Formation in the HDV Ribozyme Transition State

Journal

BIOCHEMISTRY
Volume 57, Issue 25, Pages 3465-3472

Publisher

AMER CHEMICAL SOC
DOI: 10.1021/acs.biochem.8b00031

Keywords

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Funding

  1. National Institutes of Health [1R56AI081987, 1R01AI081987, GM096000]
  2. National Institute of General Medical Sciences Medical Scientist Research Service Award [5T32 GM07281]
  3. Chemistry and Biology Interface Training Program [T32GM008720]
  4. NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R01GM096000, T32GM008720] Funding Source: NIH RePORTER

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Steric constraints imposed by the active sites of protein and RNA enzymes pose major challenges to the investigation of structure-function relationships within these systems. As a strategy to circumvent such constraints in the HDV ribozyme, we have synthesized phosphoramidites from propanediol derivatives and incorporated them at the 5'-termini of RNA and DNA oligonucleotides to generate a series of novel substrates with nucleophiles perturbed electronically through geminal fluorination. In nonenzymatic, hydroxide-catalyzed intramolecular transphosphorylation of the DNA substrates, pH-rate profiles revealed that fluorine substitution reduces the maximal rate and the kinetic pK(a) , consistent with the expected electron-withdrawing effect. In HDV ribozyme reactions, we observed that the RNA substrates undergo transphosphorylation relatively effi- ciently, suggesting that the conformational constraints imposed by a ribofuranose ring are not strictly required for ribozyme catalysis. In contrast to the nonenzymatic reactions, however, substrate fluorination modestly increases the ribozyme reaction rate, consistent with a mechanism in which (1) the 2'-hydroxyl nucleophile exists predominantly in its neutral, protonated form in the ground state and (2) the 2'-hydroxyl bears some negative charge in the rate-determining step, consistent with a transition state in which the extent of 2'-OH deprotonation exceeds the extent of P-O bond formation.

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