Kinetic and thermodynamic analysis defines roles for two metal ions in DNA polymerase specificity and catalysis

Abstract

We examined the roles of Mg2+ ions in DNA polymerization by kinetic analysis of single nucleotide incorporation catalyzed by HIV reverse transcriptase and by molecular dynamics simulation of Mg2+ binding. Binding of the Mg-nucleotide complex induces a conformational change of the enzyme from open to closed states in a process that is independent of free Mg2+ concentration. Subsequently, the second Mg2+ binds weakly to the closed state of the enzyme-DNA-Mg.dNTP complex with an apparent Kd = 3.7 mM and facilitates the catalytic reaction. This weak binding of the catalytic Mg2+ is important to maintain fidelity in that the Mg2+ samples the correctly aligned substrate without perturbing the equilibrium at physiological Mg2+ concentrations. The binding of the catalytic Mg2+ increases nucleotide specificity (kcat/Km) by increasing the rate of the chemistry and decreasing the rate of enzyme opening allowing nucleotide release. Changing the free Mg2+ concentration from 0.25 to 10 mM increased nucleotide specificity (kcat/Km) by 12-fold. Mg2+ binds very weakly to the open state of the enzyme in the absence of nucleotide (Kd {approx} 34 mM) and competes with Mg.dNTP. Analysis based on publish crystal structures showed that HIV RT binds only two metal ions during incorporation of a correct base-pair. MD simulations support the kinetic studies suggesting weak binding of the catalytic Mg2+ in open and closed states. They also support the two-metal ion mechanism, although the polymerase may bind a third metal ion in the presence of a mismatched nucleotide.