{"doi":"10.1101/2020.10.20.347138","title":"Kinetic and thermodynamic analysis defines roles for two metal ions in DNA polymerase specificity and catalysis","abstract":"Abstract We examined the roles of Mg 2+ ions in DNA polymerization by kinetic analysis of single nucleotide incorporation catalyzed by HIV reverse transcriptase and by molecular dynamics simulation of Mg 2+ 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 Mg 2+ concentration. Subsequently, the second Mg 2+ binds weakly to the closed state of the enzyme-DNA-Mg.dNTP complex with an apparent K d = 3.7 mM and facilitates the catalytic reaction. This weak binding of the catalytic Mg 2+ is important to maintain fidelity in that the Mg 2+ samples the correctly aligned substrate without perturbing the equilibrium at physiological Mg 2+ concentrations. The binding of the catalytic Mg 2+ increases nucleotide specificity ( k cat / K m ) by increasing the rate of the chemistry and decreasing the rate of enzyme opening allowing nucleotide release. Changing the free Mg 2+ concentration from 0.25 to 10 mM increased nucleotide specificity ( k cat / K m ) by 12-fold. Mg 2+ binds very weakly to the open state of the enzyme in the absence of nucleotide ( K d ≈ 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 Mg 2+ 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.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2020,"id":127539,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9476,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2020-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":376986,"name":"Serdal Kırmızıaltın","orcid":"0000-0001-8380-5725","position":1,"is_corresponding":false},{"id":376987,"name":"Adrienne Chang","orcid":"0000-0003-3497-1561","position":2,"is_corresponding":false},{"id":376988,"name":"Joshua E. Mayfield","orcid":"0000-0002-5253-0084","position":3,"is_corresponding":false},{"id":348305,"name":"Yan Zhang","orcid":"0000-0002-9360-5388","position":4,"is_corresponding":false},{"id":348304,"name":"Kenneth A. Johnson","orcid":"0000-0002-6575-2823","position":5,"is_corresponding":false},{"id":376985,"name":"Shanzhong Gong","orcid":"0000-0001-8750-9907","position":0,"is_corresponding":true}],"reference_count":53,"raw_metadata":null,"created_at":"2026-07-18T23:15:34.966380Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}