Determination of hepatitis delta virus ribozyme N(-1) nucleobase and functional group specificity using internal competition kinetics.

Biological catalysis involves interactions distant from the site of chemistry that can position the substrate for reaction. Catalysis of RNA 2'-O-transphosphorylation by the hepatitis delta virus (HDV) ribozyme is sensitive to the identity of the N(-1) nucleotide flanking the reactive phosphoryl group. However, the interactions that affect the conformation of this position, and in turn the 2'O nucleophile, are unclear.

Altered (transition) states: mechanisms of solution and enzyme catalyzed RNA 2'-O-transphosphorylation.

Although there have been great strides in defining the mechanisms of RNA strand cleavage by 2'-O-transphosphorylation, long-standing questions remain. How do different catalytic modes such as acid/base and metal ion catalysis influence transition state charge distribution? Does the large rate enhancement characteristic of biological catalysis result in different transition states relative to solution reactions? Answering these questions is important for understanding biological catalysis in general, and revealing principles for designing small molecule inhibitors. Recent application of linear free energy relationships and kinetic isotope effects together with multi-scale computational simulations are providing tentative answers to these questions for this fundamentally important class of phosphoryl transfer reactions.

Experimental and computational analysis of the transition state for ribonuclease A-catalyzed RNA 2'-O-transphosphorylation.

Enzymes function by stabilizing reaction transition states; therefore, comparison of the transition states of enzymatic and nonenzymatic model reactions can provide insight into biological catalysis. Catalysis of RNA 2'-O-transphosphorylation by ribonuclease A is proposed to involve electrostatic stabilization and acid/base catalysis, although the structure of the rate-limiting transition state is uncertain. Here, we describe coordinated kinetic isotope effect (KIE) analyses, molecular dynamics simulations, and quantum mechanical calculations to model the transition state and mechanism of RNase A.

Kinetic isotope effects for RNA cleavage by 2'-O- transphosphorylation: nucleophilic activation by specific base.

To better understand the interactions between catalysts and transition states during RNA strand cleavage, primary (18)O kinetic isotope effects (KIEs) and solvent D(2)O isotope effects were measured to probe the mechanism of base-catalyzed 2'-O-transphosphorylation of the RNA dinucleotide 5'-UpG-3'. The observed (18)O KIEs for the nucleophilic 2'-O and in the 5'-O leaving group at pH 14 are both large relative to reactions of phosphodiesters with good leaving groups, indicating that the reaction catalyzed by hydroxide has a transition state (TS) with advanced phosphorus-oxygen bond fission to the leaving group ((18)k(LG) = 1.034 +/- 0.