The nucleic acids RNA and DNA consist of nucleoside building blocks joined by phosphodiester linkages. Phosphodiesters are generally inert to hydrolytic cleavage under physiological conditions because their negative charge disfavors nucleophilic attack. However, the hydrolytic scission of phosphodicster linkages is an important and common biological process, and can occur rapidly in the presence of appropriate catalysts such as ribozymes and nuclease enzymes. Metals play an important role in this process. Several possible modes of action can bc invoked for metal-promoted phosphate ester hydrolysis, including Lewis acid catalysis, Bronsted base catalysis by metal-bound hydroxides, nucleophilic catalysis by metal-bound hydroxides, Brmsted acid catalysis by metal-bound water, and electrostatic stabilization of transition states by positively charged metal ions. Here we critically discuss the roles of metals in the hydrolytic cleavage of nucleic acids and related model substrates.
The cleavage (transesterification) of polyribonucleotides is a
process of considerable interest. The use of
dinucleotide RNA fragments as substrates for the screening of RNA
catalysis agents and mechanistic studies is
widespread. This practice may not accurately predict the relative
abilities of metal complexes to cleave
polyribonucleotide substrates. We report the use of chimeric
DNA/RNA molecules, containing RNA nucleotides
embedded in DNA sequences, as substrates for studying the
transesterification of RNA. The substrates, termed
embRNA, display the simplicity of dinucleotide substrates while
possessing the multiple phosphate and nucleobase
metal-binding sites found in polyribonucleotides. In addition, the
DNA residues provide an internal check for oxidative
cleavage. The synthesis, purification, and activity of our
first-generation embRNA, T11UT7A, is
described. T11UT7A is a substrate for the ribonuclease RNase 1, and RNase
1 cleavage provides an excellent measure of the extent
of 2‘-deprotection in the synthetic embRNA. Cleavage of
T11UT7A by hydroxide and a variety of
metal ions and
complexes is also reported, and the use of embRNA in kinetic assays is
demonstrated. Competitive cleavage of
RNA and DNA is built into the embRNA assay. With Pb(II),
Ce(III), and Cu(II) reagents, we observed
efficient
RNA cleavage and no DNA cleavage. Kinetic comparison is made
between embRNA T11UT7 and the
analogous,
all-RNA substrate U19.
Aqua( 2.2': 6',2"-terpyridine)copper( 11) catalyses the hydrolysis of 2',3'-cyclic adenosine monophosphate, but does not hydrolyse the 'activated' substrate bis(p-nitrophenyl) phosphate indicating that the latter is not a reliable model for biological phosphoric ester hydrolysis.
The hydrolysis and transesterification of RNA are catalyzed by a variety of metal ions, metal complexes,
metalloenzymes, and ribozymes. These reactions are of fundamental biochemical importance. However, the role
that metal ions play in RNA hydrolysis is not completely understood. We previously showed that aqueous Cu(II)
terpyridine (Cutrpy) is effective for both transesterification and hydrolysis of RNA, and we harnessed this reactivity
by constructing ribozyme mimics that employ terpyridyl Cu(II) in their active sites. Here we report a detailed
kinetic study of the hydrolysis of adenosine-2‘,3‘-cyclic monophosphate (cAMP) by Cutrpy. The reaction is
established to be first-order in Cutrpy and first-order in substrate. Catalytic turnover is observed, although product
inhibition occurs. Chloride ion also inhibits the reaction, which indicates the disadvantage of using NaCl as an
ionic strength buffer in related studies. The pH−rate profile is sigmoidal and implicates the hydroxide form of
the catalyst, CutrpyOH+, as the active species. Isotope effects were used to determine whether the metal hydroxide
acts as a nucleophile or a base. The solvent deuterium kinetic isotope effect, k
H/k
D, was measured to be 1.0 ± 0.1
after considering equilibrium isotope effects. To assist the mechanistic interpretation of the measured isotope
effects, the fractionation factors for a dianionic phosphorane transition state (methyl phosphate dianion) and
hydroxide were evaluated by ab initio quantum chemical calculations. Considering the calculated results along
with the relevant experimental fractionation factors, isotope effects were predicted for three overall mechanisms:
nucleophilic catalysis, general base catalysis, and specific base/general acid catalysis. We conclude that CutrpyOH+
acts as a nucleophilic catalyst in the hydrolysis of cAMP. This contrasts with the behavior of CutrpyOH+ as a
base catalyst in the transesterification of RNA.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.