{"doi":"10.1021/jacs.5c00521","title":"Transition State Analysis of SAMHD1 from Primary <sup>18</sup>O, <sup>33</sup>P, and Solvent Kinetic Isotope Effects","abstract":"Human sterile alpha motif and HD-domain-containing protein 1 (SAMHD1) is an allosterically regulated dNTP triphosphohydrolase (dNTP + H 2 O → dNuc + PPPi) involved in deoxynucleotide regulation and DNA repair. We characterized the chemical features of the SAMHD1 transition state for 2′-deoxyadenosine 5′-triphosphate (dATP) hydrolysis by analysis of 18 O and 33 P primary kinetic isotope effects (KIEs) at the α-phosphoryl of the leaving triphosphate group. The intrinsic KIE values for [5′- 18 O]dATP of 1.028 ± 0.003 and for [α- 33 P]dATP of 1.015 ± 0.004 provide insights into the mechanistic details of the SAMHD1 transition state. Solvent 2 H 2 O isotope effects for the hydrolysis of dATP indicate that a single proton is being transferred at the transition state to give a solvent KIE of 3.2 ± 0.1. Quantum chemical matching of the isotope effects supports a concerted, loose, highly asymmetric D N A N transition state with a Pauling bond order of 0.17 to the attacking hydroxide oxygen nucleophile and 0.53 to the departing deoxyadenosine. The reaction coordinate distance is 4.7 Å from attacking the hydroxyl oxygen to departing 5′-deoxyadenosine oxygen. The solvent KIE is consistent with a near-midpoint proton transfer from the His215 catalytic site proton donor to the deoxyadenosine 5′-oxygen in the transition state. This is the first triphosphohydrolase transition state to be characterized and the first use of a 33 P primary isotope effect to characterize a phosphotransferase transition state.","journal":"Journal of the American Chemical Society","year":2025,"id":532017,"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":3,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9535,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1413267,"name":"D. Groom","orcid":null,"position":1,"is_corresponding":false},{"id":369385,"name":"Vern L. Schramm","orcid":"0000-0002-8056-1929","position":2,"is_corresponding":false},{"id":1412798,"name":"Ananda K. Ghosh","orcid":"0000-0002-5485-4760","position":0,"is_corresponding":true}],"reference_count":54,"raw_metadata":null,"created_at":"2026-07-19T02:51:18.928280Z","pmid":"40014869","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":[]}