What is the relationship between polymerase nuclotide incorporation rate and fidelity?

Abstract

Our laboratory has employed the RNA-dependent RNA polymerase from poliovirus (3Dpol) as a model system to study the chemistry of nucleotidyl transfer. Solvent deuterium kinetic isotope effect and proton inventory studies have shown two proton transfer reactions in the rate-limiting transition state for nucleotidyl transfer. We have identified a lysine residue (Lys-359) in the polymerase active site that protonates the pyrophosphate (PPi) leaving group during nucleotidyl transfer. Because both Lys-359 and PPi have pKa value in the 9–10 regime, proton transfer between these molecules should be maximized. However, nucleotidyl transfer efficiency should be diminished by changing Lys-359 to His, Arg or Leu due to differences in pKa values or the inability to serve as a proton donor. We show that these substitutions reduce elongation rate by 10–50 fold relative to wild-type enzyme. Loss of a proton donor (Leu-359 3Dpol) yields an enzyme that is incapable of nucleotide misincorporation. The fidelity of Arg-359 3Dpol was equivalent to WT 3Dpol; however, the fidelity of His-359 3Dpol was more stringent than WT 3Dpol. These data suggest that polymerase fidelity is not governed by the overall observed rate of nucleotide incorporation. We suggest that the extent to which chemistry contributes to the overall rate of nucleotide incorporation may influence fidelity. This research is supported by a grant (AI45818) from the NIAID/NIH.