Computer simulation of the chemical catalysis of DNA polymerases: discriminating between alternative nucleotide insertion mechanisms for T7 DNA polymerase.

摘要: Understanding the chemical step in catalytic reaction of DNA polymerases is essential for elucidating molecular basis fidelity replication. The present work evaluates free energy surface nucleotide transfer T7 polymerase by perturbation/empirical valence bond (FEP/EVB) calculations. A key aspect enzyme simulation a comparison enzymatic profiles with corresponding reference reactions water using same computational methodology, thereby enabling quantitative estimate insertion reaction. driven FEP/EVB methodology between structures representing reactant, pentacovalent intermediate, and product states. This pathway corresponds to three microscopic steps, deprotonation attacking group, nucleophilic attack on Pα atom dNTP substrate, departure leaving group. Three different mechanisms first...