Department of Chemistry and Biochemistry, 1068 W Sheridan Rd, Loyola University Chicago, Chicago, IL, 60660, USA.
Department of Chemistry and Biochemistry, 1068 W Sheridan Rd, Loyola University Chicago, Chicago, IL, 60660, USA.
Arch Biochem Biophys. 2024 Nov;761:110162. doi: 10.1016/j.abb.2024.110162. Epub 2024 Sep 23.
Thioredoxin/glutathione reductase from Schistosoma mansoni (SmTGR) is a multifunctional enzyme that catalyzes the reduction of glutathione (GSSG) and thioredoxin, as well as the deglutathionylation of peptide and non-peptide substrates. SmTGR structurally resembles known glutathione reductases (GR) and thioredoxin reductases (TrxR) but with an appended N-terminal domain that has a typical glutaredoxin (Grx) fold. Despite structural homology with known GRs, the site of GSSG reduction has frequently been reported as the Grx domain, based primarily on aerobic, steady-state kinetic measurements and x-ray crystallography. Here, we present an anaerobic characterization of a series of variant SmTGRs to establish the site of GSSG reduction as the cysteine pair most proximal to the FAD, Cys154/Cys159, equivalent to the site of GSSG reduction in GRs. Anaerobic steady-state analysis of U597C, U597S, U597C + C31S, and I592STOP SmTGR demonstrate that the Grx domain is not involved in the catalytic reduction of GSSG, as redox silencing of the C-terminus results in no modulation of the observed turnover number (∼0.025 s) and redox silencing of the Grx domain results in an increased observed turnover number (∼0.08 s). Transient-state single turnover analysis of these variants corroborates this, as the slowest rate observed titrates hyperbolically with GSSG concentration and approaches a limit that coincides with the respective steady-state turnover number for each variant. Numerical integration fitting of the transient state data can only account for the observed trends when competitive binding of the C-terminus is included, indicating that the partitioning of electrons to either substrate occurs at the Cys154/Cys159 disulfide rather than the previously proposed Cys596/Sec597 sulfide/selenide. Paradoxically, truncating the C-terminus at Ile592 results in a loss of GR activity, indicating a crucial non-redox role for the C-terminus.
曼氏血吸虫硫氧还蛋白/谷胱甘肽还原酶（SmTGR）是一种多功能酶，可催化谷胱甘肽（GSSG）和硫氧还蛋白的还原，以及肽和非肽底物的去谷胱甘肽化。SmTGR 的结构与已知的谷胱甘肽还原酶（GR）和硫氧还蛋白还原酶（TrxR）相似，但具有一个附加的 N 端结构域，该结构域具有典型的谷氧还蛋白（Grx）折叠。尽管与已知的 GR 具有结构同源性，但 GSSG 还原的部位经常被报道为 Grx 结构域，这主要基于需氧、稳态动力学测量和 X 射线晶体学。在这里，我们对一系列 SmTGR 变体进行了厌氧特性分析，以确定 GSSG 还原的部位是最靠近 FAD 的半胱氨酸对，即 Cys154/Cys159，与 GR 中 GSSG 还原的部位相当。U597C、U597S、U597C+C31S 和 I592STOP SmTGR 的厌氧稳态分析表明，Grx 结构域不参与 GSSG 的催化还原，因为 C 端的氧化还原沉默不会导致观察到的周转率（约 0.025 s）发生调制，而 Grx 结构域的氧化还原沉默会导致观察到的周转率增加（约 0.08 s）。这些变体的瞬态单转换分析证实了这一点，因为观察到的最慢速率与 GSSG 浓度呈双曲线滴定，并接近每个变体的相应稳态周转率的极限。瞬态数据的数值积分拟合只能在包含 C 端竞争性结合时才能解释观察到的趋势，这表明电子分配到任一底物都发生在 Cys154/Cys159 二硫键上，而不是之前提出的 Cys596/Sec597 硫/硒化物。矛盾的是，在 Ile592 处截断 C 端会导致 GR 活性丧失，表明 C 端具有至关重要的非氧化还原作用。
