| Autor: |
Gogar RK; Department of Chemistry & Biochemistry, The University of Alabama, Tuscaloosa, Alabama 35487, United States., Frantom PA; Department of Chemistry & Biochemistry, The University of Alabama, Tuscaloosa, Alabama 35487, United States. |
| Jazyk: |
angličtina |
| Zdroj: |
Biochemistry [Biochemistry] 2024 Jun 18; Vol. 63 (12), pp. 1569-1577. Date of Electronic Publication: 2024 May 30. |
| DOI: |
10.1021/acs.biochem.4c00084 |
| Abstrakt: |
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. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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