Wang Shuping, Geng Zhirong, Shi Nan, Li Xiangli, Wang Zhilin
State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, Jiangsu, PR China.
PLoS One. 2014 Oct 28;9(10):e110924. doi: 10.1371/journal.pone.0110924. eCollection 2014.
Arsenic (III) methyltransferase (AS3MT) is a cysteine (Cys)-rich enzyme that catalyzes the biomethylation of arsenic. To investigate how these crucial Cys residues promote catalysis, we used matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) to analyze Cys residues in recombinant human arsenic (III) methyltransferase (hAS3MT). We detected two disulfide bonds, Cys250-Cys32 and Cys368-Cys369, in hAS3MT. The Cys250-Cys32 disulfide bond was reduced by glutathione (GSH) or other disulfide bond reductants before the enzymatic methylation of arsenite (iAs3+). In addition to exposing residues around the active sites, cleavage of the Cys250-Cys32 pair modulated the conformation of hAS3MT. This adjustment may stabilize the binding of S-Adenosyl-L-methionine (AdoMet) and favor iAs3+ binding to hAS3MT. Additionally, we observed the intermediate of Cys250-S-adenosylhomocysteine (AdoHcy), suggesting that Cys250 is involved in the transmethylation. In recovery experiments, we confirmed that trivalent arsenicals were substrates for hAS3MT, methylation of arsenic occurred on the enzyme, and an intramolecular disulfide bond might be formed after iAs3+ was methylated to dimethylarsinous acid (DMA3+). In this work, we clarified both the functional roles of GSH and the crucial Cys residues in iAs3+ methylation catalyzed by hAS3MT.
砷（III）甲基转移酶（AS3MT）是一种富含半胱氨酸（Cys）的酶，可催化砷的生物甲基化。为了研究这些关键的半胱氨酸残基如何促进催化作用，我们使用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）分析重组人砷（III）甲基转移酶（hAS3MT）中的半胱氨酸残基。我们在hAS3MT中检测到两个二硫键，即Cys250-Cys32和Cys368-Cys369。在亚砷酸盐（iAs3+）进行酶促甲基化之前，Cys250-Cys32二硫键可被谷胱甘肽（GSH）或其他二硫键还原剂还原。除了暴露活性位点周围的残基外，Cys250-Cys32对的裂解还调节了hAS3MT的构象。这种调节可能会稳定S-腺苷-L-甲硫氨酸（AdoMet）的结合，并有利于iAs3+与hAS3MT的结合。此外，我们观察到了Cys250- S-腺苷同型半胱氨酸（AdoHcy）中间体，表明Cys250参与了转甲基作用。在回收实验中，我们证实三价砷化合物是hAS3MT的底物，砷在酶上发生甲基化，并且在iAs3+甲基化为二甲基亚胂酸（DMA3+）后可能形成分子内二硫键。在这项工作中，我们阐明了GSH的功能作用以及hAS3MT催化iAs3+甲基化过程中关键的半胱氨酸残基的作用。
