title: Kinetic analysis of the bisubstrate cysteine desulfurase SufS from Bacillus subtilis.
abstract: Cysteine is the major sulfur donor for thio cofactors in bacterial and eukaryotic systems. The first step in sulfur mobilization involves a PLP-dependent enzymatic mechanism. During catalysis, free cysteine is converted into alanine with the concomitant formation of a persulfide bond with the catalytic cysteine residue, thus forming a covalent enzyme intermediate. Cysteine desulfurases in their persulfurated forms serve as donors at the intersection of various cellular sulfur-requiring pathways. Most Gram-positive bacteria, including Bacillus subtilis, contain a cysteine desulfurase gene sufS located adjacent to the gene encoding the proposed Fe-S cluster scaffold SufU. In this work, we identified the participation of SufU as a substrate in the SufS catalytic mechanism. Development of a sensitive method for detection of alanine formed in the SufS reaction enabled the identification of its associated mechanistic features. Steady-state kinetic analysis of alanine formation provided evidence of a double-displacement mechanism (ping-pong) of the cysteine:SufU sulfurtransferase reaction catalyzed by SufS. Results from site-directed mutagenesis of the catalytic cysteine (SufS(C361A)) and iodoacetamide alkylation of SufU support the occurrence of persulfide sulfur transfer steps in the mechanism of SufS.
authors.affiliation: Department of Chemistry, Wake Forest University, Winston-Salem, North Carolina 27109-7486, USA.
authors.forename: Bruna
authors.initials: B
authors.lastname: Selbach
authors.forename: Emily
authors.initials: E
authors.lastname: Earles
authors.forename: Patricia C
authors.initials: PC
authors.lastname: Dos Santos
mesh_terms: D000409:Alanine / Q000378:metabolism; D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000378:metabolism*; D013437:Carbon-Sulfur Lyases / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000378:metabolism*; D007700:Kinetics
chemical_list: D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; D013437:Carbon-Sulfur Lyases; C080546:cysteine desulfurase; D003545:Cysteine; D000409:Alanine