Kinetic and thermodynamic analysis defines roles for two metal ions in DNA polymerase specificity and catalysis.
Magnesium ions play a critical role in catalysis by many enzymes and contribute to the fidelity of DNA polymerases through a two-metal ion mechanism. However, specificity is a kinetic phenomenon and the roles of Mg2+ ions in each step in the catalysis have not been resolved. We first examined the roles of Mg2+ by kinetic analysis of single nucleotide incorporation catalyzed by HIV reverse transcriptase. We show that Mg.dNTP binding induces an enzyme conformational change at a rate that is independent of free Mg2+ concentration. Subsequently, the second Mg2+ binds to the closed state of the enzyme-DNA-Mg.dNTP complex (Kd = 3.7 mM) to facilitate catalysis. Weak binding of the catalytic Mg2+ contributes to fidelity by sampling the correctly aligned substrate without perturbing the equilibrium for nucleotide binding at physiological Mg2+ concentrations. An increase of the Mg2+ concentration from 0.25 to 10 mM increases nucleotide specificity (kcat/Km) 12-fold largely by increasing the rate of the chemistry relative to the rate of nucleotide release. Mg2+ binds very weakly (Kd ≤ 37 mM) to the open state of the enzyme. Analysis of published crystal structures showed that HIV reverse transcriptase binds only two metal ions prior to incorporation of a correct base pair. Molecular dynamics simulations support the two-metal ion mechanism and the kinetic data indicating weak binding of the catalytic Mg2+. Molecular dynamics simulations also revealed the importance of the divalent cation cloud surrounding exposed phosphates on the DNA. These results enlighten the roles of the two metal ions in the specificity of DNA polymerases.
['Gong|Shanzhong|S|', 'Kirmizialtin|Serdal|S|', 'Chang|Adrienne|A|', 'Mayfield|Joshua E|JE|', 'Zhang|Yan Jessie|YJ|', 'Johnson|Kenneth A|KA|']
D002413:Cations, Divalent D015658:HIV Infections D054303:HIV Reverse Transcriptase D015497:HIV-1 D006801:Humans D007700:Kinetics D008274:Magnesium D056004:Molecular Dynamics Simulation D011487:Protein Conformation D013816:Thermodynamics
metal ions magnesium conformational change molecular dynamics DNA replication enzyme catalysis enzyme specificity induced fit transient kinetics