Mechanistic cross-talk between DNA/RNA polymerase enzyme kinetics and nucleotide substrate availability in cells: Implications for polymerase inhibitor discovery

Abstract

Enzyme kinetic analysis reveals a dynamic relationship between enzymes and their substrates. Overall enzyme activity can be controlled by both protein expression and various cellular regulatory systems. Interestingly, the availability and concentrations of intracellular substrates can constantly change, depending on conditions and cell types. Here, we review previously reported enzyme kinetic parameters of cellular and viral DNA and RNA polymerases with respect to cellular levels of their nucleotide substrates. This broad perspective exposes a remarkable co-evolution scenario of DNA polymerase enzyme kinetics with dNTP levels that can vastly change, depending on cell proliferation profiles. Similarly, RNA polymerases display much higher K_m values than DNA polymerases, possibly due to millimolar range rNTP concentrations found in cells (compared with micromolar range dNTP levels). Polymerases are commonly targeted by nucleotide analog inhibitors for the treatments of various human diseases, such as cancers and viral pathogens. Because these inhibitors compete against natural cellular nucleotides, the efficacy of each inhibitor can be affected by varying cellular nucleotide levels in their target cells. Overall, both kinetic discrepancy between DNA and RNA polymerases and cellular concentration discrepancy between dNTPs and rNTPs present pharmacological and mechanistic considerations for therapeutic discovery.

Keywords: DNA polymerase; RNA polymerase; dNTP; enzyme inhibitor; enzyme kinetics; inhibitors; nucleoside/nucleotide metabolism; rNTP.

Figure 1.

Comparison of DNA and RNA polymerase steady-state kinetic activity and correlation with intracellular dNTP and rNTP concentrations. The combined human replicative DNA polymerases (α, δ, and ε) and HIV-1 RT K_m values (expressed as ranges consisting of reported mean K_m values, as described in Table 1) are denoted in reference to the plotted mean intracellular dNTPs found in dividing cells and nondividing cells (human monocyte–derived macrophages). rNTP concentrations found to be relatively high across cell types (20, 25) were plotted to be compared with the range of high rNTP concentrations required for initiation of viral RdRP polymerization described in text (Flaviviridae RdRP kinetics section) (107–109, 113, 114, 130, 131).

Figure 2.

Connections among nucleotide regulation status, cellular nucleotide levels, and enzyme kinetic values of polymerases. Ranges of steady-state kinetic K_m values of DNA and RNA polymerases (orange) are arranged relative to cellular concentrations of their nucleotide substrates (blue). Intracellular nucleotide concentrations vary among cell types and are dependent upon differential metabolic regulations (green).