{"doi":"10.1101/2020.12.21.423855","title":"HIV reverse transcriptase pre-steady-state kinetic analysis of chain terminators and translocation inhibitors reveals interactions between magnesium and nucleotide 3′-OH","abstract":"Abstract Deoxythymidine triphosphate analogs with various 3′ sugar groups (-OH (dTTP), -H, -N 3 , -NH 2 , -F, -O-CH 3 , no group (2′,3′-didehydro-2′,3′-dideoxythymidine triphosphate (d4TTP)), and those retaining the 3′-OH but with 4′ additions (4′-C-methyl, 4′-C-ethyl) or sugar ring modifications (D-carba dTTP) were evaluated using pre-steady-state kinetics in low (0.5 mM) and high (6 mM) Mg 2+ with HIV reverse transcriptase (RT). Analogs showed diminished incorporation rates ( k ) compared to dTTP ranging from about 2-fold (3′-H, -N 3 , and d4TTP with high Mg 2+ ) to >10-fold (3′-NH 2 and 3′-F with low Mg 2+ ), while 3′-O-CH 3 dTTP incorporated much slower than other analogs. Illustrating the importance of interactions between Mg 2+ and the 3′-OH, k using 5 μM dTTP and 0.5 mM Mg 2+ was only modestly slower (1.6-fold) than with 6 mM Mg 2+ , while analogs with 3′ alterations incorporated 2.8-5.1-fold slower in 0.5 mM Mg 2+ . In contrast, 4′-C-methyl and D-carb dTTP, which retain the 3′-OH, were not significantly affected by Mg 2+ . Consistent with these results, analogs with 3′ modifications were better inhibitors in 6 mM vs . 0.5 mM Mg 2+ . Equilibrium dissociation constant (K d ) and k pol determinations for dTTP and analogs lacking a 3′-OH indicated that low Mg 2+ caused a several-fold greater reduction in k pol with the analogs but had little effect on K d , results consistent with a role for 3′-OH/Mg 2+ interactions in catalysis rather than nucleotide binding. Overall, results emphasize the importance previously unreported interactions between Mg 2+ and the 3′-OH of the incoming nucleotide and suggest inhibitors with 3′-OH groups may have advantages in the low free Mg 2+ in physiological settings.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2020,"id":132048,"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":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.958,"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":375980,"name":"Jeffrey J. DeStefano","orcid":"0000-0002-8710-7622","position":1,"is_corresponding":false},{"id":587498,"name":"Christopher R. Dilmore","orcid":null,"position":0,"is_corresponding":true}],"reference_count":42,"raw_metadata":null,"created_at":"2026-07-18T23:16:07.542484Z","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":[]}