Abstract:
Hydrogen sulfide (H2S) is a signaling molecule with many beneficial effects. However, its cellular concentration is strictly regulated to avoid toxicity. Persulfide dioxygenase (PDO or ETHE1) is a mononuclear non-heme iron-containing protein in the sulfide oxidation pathway catalyzing the conversion of GSH persulfide (GSSH) to sulfite and GSH. PDO mutations result in the autosomal-recessive disorder ethylmalonic encephalopathy (EE). Here, we developed γ-glutamyl-homocysteinyl-glycine (GHcySH), in which the cysteinyl moiety in GSH is substituted with homocysteine, as a mechanism-based PDO inhibitor. Human PDO used GHcySH as an alternative substrate and converted it to GHcy-SO2H, mimicking GS-SO2H, the putative oxygenated intermediate formed with the natural substrate. Because GHcy-SO2H contains a C-S bond rather than an S-S bond in GS-SO2H, it failed to undergo the final hydrolysis step in the catalytic cycle, leading to PDO inhibition. We also characterized the biochemical penalties incurred by the L55P, T136A, C161Y, and R163W mutations reported in EE patients. The variants displayed lower iron content (1.4-11-fold) and lower thermal stability (1.2-1.7-fold) than WT PDO. They also exhibited varying degrees of catalytic impairment; the kcat/Km values for R163W, L55P, and C161Y PDOs were 18-, 42-, and 65-fold lower, respectively, and the T136A variant was most affected, with a 200-fold lower kcat/Km Like WT enzyme, these variants were inhibited by GHcySH. This study provides the first characterization of an intermediate in the PDO-catalyzed reaction and reports on deficits associated with EE-linked mutations that are distal from the active site.
摘要:
硫化氢(H2S)是具有许多有益作用的信号分子。然而,其细胞浓度受到严格管制以避免毒性。过硫化物双加氧酶(PDO或ETHE1)是硫化物氧化途径中的单核非血红素含铁蛋白,催化GSH过硫化物(GSSH)转化为亚硫酸盐和GSH。PDO突变导致常染色体隐性遗传疾病乙基丙二酸脑病(EE)。这里,我们开发了γ-谷氨酰-同型半胱氨酸-甘氨酸(GHcySH),其中GSH中的半胱氨酰部分被高半胱氨酸取代,作为一种基于机制的PDO抑制剂。人类PDO使用GHcySH作为替代底物,并将其转化为GHcy-SO2H,模仿GS-SO2H,与天然底物形成的推定的含氧中间体。由于GHcy-SO2H包含C-S键而不是GS-SO2H中的S-S键,它未能在催化循环中经历最终的水解步骤,导致PDO抑制。我们还描述了L55P引起的生化惩罚,T136A,C161Y,在EE患者中报告了R163W突变。变体显示出比WTPDO更低的铁含量(1.4-11倍)和更低的热稳定性(1.2-1.7倍)。它们还表现出不同程度的催化损害;R163W的kcat/Km值,L55P,C161YPDO为18-,42-,低65倍,分别,T136A变种受影响最大,与WT酶一样,kcat/Km降低200倍,这些变体被GHcySH抑制.这项研究提供了PDO催化反应中中间体的第一个表征,并报告了与活性位点远离EE连接的突变相关的缺陷。