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

ABSTRACT: 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 (K_d = 3.7 mM) to facilitate catalysis. Weak binding of the catalytic Mg²⁺ contributes to fidelity by sampling

SUBMITTER: Gong S 

PROVIDER: S-EPMC7948414 | biostudies-literature | 2021 Jan-Jun

REPOSITORIES: biostudies-literature