Department of Chemistry, Wake Forest University, Winston-Salem, North Carolina 27109-7486, USA.
Biochemistry. 2010 Oct 12;49(40):8794-802. doi: 10.1021/bi101358k. Epub 2010 Sep 16.
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.
半胱氨酸是细菌和真核生物系统中硫代辅因子的主要硫供体。硫动员的第一步涉及一个依赖于 PLP 的酶促机制。在催化过程中，游离半胱氨酸转化为丙氨酸，同时与催化半胱氨酸残基形成过硫键，从而形成共价酶中间物。过硫化形式的半胱氨酸脱硫酶作为各种细胞硫需求途径的交点的供体。大多数革兰氏阳性菌，包括枯草芽孢杆菌，都含有一个位于拟铁-硫簇支架 SufU 编码基因旁边的半胱氨酸脱硫酶基因 sufS。在这项工作中，我们确定了 SufU 作为 SufS 催化机制中的底物参与。开发一种灵敏的方法来检测 SufS 反应中形成的丙氨酸，使我们能够鉴定其相关的机制特征。丙氨酸形成的稳态动力学分析为 SufS 催化的半胱氨酸：SufU 硫转移酶反应的双置换机制（乒乓）提供了证据。对催化半胱氨酸（SufS(C361A)）的定点突变和 SufU 的碘乙酰胺烷基化的结果支持了 SufS 机制中过硫化硫转移步骤的发生。
