{"doi":"10.1021/acs.biochem.4c00084","title":"Persulfide Transfer to SufE Activates the Half-Sites Reactivity of the <i>E. coli</i> Cysteine Desulfurase SufS","abstract":"The Escherichia coli cysteine desulfurase SufS (EcSufS) is a dimeric, PLP-dependent enzyme responsible for sulfur mobilization in the SUF Fe–S cluster bioassembly pathway. The enzyme uses cysteine as a sulfur source and generates alanine and a covalent persulfide located on an active site of cysteine. Optimal in vitro activity of EcSufS requires the presence of the transpersulfurase protein, EcSufE, and a strong reductant. Here, presteady-state and single-turnover kinetics are used to investigate the mechanism of EcSufS activation by EcSufE. In the absence of EcSufE, EcSufS exhibits a presteady-state burst of product production with an amplitude of ∼0.4 active site equivalents, consistent with a half-sites reactivity. KinTek Explorer was used to isolate the first turnover of alanine formation and fit the data with a simplified kinetic mechanism with steps for alanine formation ( k 3 ) and a net rate constant for the downstream steps ( k 5 ). Using this treatment, microscopic rate constants of 2.3 ± 0.5 s –1 and 0.10 ± 0.01 s –1 were determined for k 3 and k 5, respectively. The inclusion of EcSufE in the reaction results in a similar rate constant for k 3 but induces a 10-fold enhancement of k 5 to 1.1 ± 0.2 s –1, such that both steps are partially rate-determining. The most likely downstream step where EcSufE could exert influence on EcSufS activity is the removal of the persulfide intermediate. Importantly, this step appears to serve as a limiting feature in the half-sites activity such that activating persulfide transfer allows for rapid shifting between active sites. Single-turnover assays show that the presence of EcSufE slightly slowed the rates of alanine-forming steps, suggesting it does not activate steps in the desulfurase half reaction.","journal":"Biochemistry","year":2024,"id":457718,"datarank":0.26876392038420827,"base_score":1.791759469228055,"endowment":1.791759469228055,"self_citation_contribution":0.26876392038420827,"citation_network_contribution":0.0,"self_endowment_contribution":0.26876392038420827,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":5,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9569,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":440448,"name":"Patrick A. Frantom","orcid":"0000-0001-7655-5967","position":1,"is_corresponding":false},{"id":1072839,"name":"Rajleen K. Gogar","orcid":"0000-0002-5032-5749","position":0,"is_corresponding":true}],"reference_count":19,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T02:03:41.744499Z","pmid":"38813769","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":[]}