Abstract
In the human body, arsenic is metabolized by methylation. Understanding this process is important and provides insight into the relationship between arsenic and its related diseases. We used the rapid equilibrium kinetic model to study the reaction sequence of arsenite methylation. The results suggest that the mechanism for arsenite methylation is a completely ordered mechanism that is also of general interest in reaction systems with different reductants, such as tris(2-carboxyethyl)phosphine, cysteine, and glutathione. In the reaction, cysteine residues of recombinant human arsenic (+3 oxidation state) methyltransferase (hAS3MT) coordinate with arsenicals and involve the methyl transfer step. S-Adenosyl-l-methionine (AdoMet) is the first-order reactant, which modulates the conformation of hAS3MT to a best matched state by hydrophobic interaction. As the second-order reactant, reductant reduces the disulfide bond, most likely between Cys-250 and another cysteine residue of hAS3MT, and exposes the active site cysteine residues for binding trivalent inorganic arsenic (iAs(3+)) to give monomethylarsonic dicysteine (MADC(3+)). In addition, the reaction can be extended to further methylate MADC(3+) to dimethylarsinic cysteine (DAMC(3+)). In the methylation reaction, the β-pleated sheet content of hAS3MT is increased, and the hydrophobicity of the microenvironment around the active sites is decreased. Similarly, we confirm that both the high β-pleated sheet content of hAS3MT and the high dissociation ability of the enzyme-AdoMet-reductant improve the yield of dimethylated arsenicals.
Highlights
Oxidative methylation and successive methylation are two possible enzymatic mechanisms of arsenite methylation
To elucidate the detailed mechanism, we studied the involvement of the cysteine residue of human Arsenic (ϩ3 oxidation state) methyltransferase (AS3MT) in the methylation reaction by site-directed mutagenesis and found that the conserved Cys-156 and Cys-206 were essential for catalytic activity and that Cys-72 and Cys-250 played a key role in the reaction [20, 26]
Our results suggest that the valence state of arsenic is unchanged when the methyl transfer step occurs on hAS3MT
Summary
Oxidative methylation and successive methylation are two possible enzymatic mechanisms of arsenite methylation. Cysteine residues of recombinant human arsenic (؉3 oxidation state) methyltransferase (hAS3MT) coordinate with arsenicals and involve the methyl transfer step. The formation of the As-glutathione (GSH) complex was considered to be essential for each of the methylation steps, and the intermediates were arsenic triglutathione (ATG3ϩ), monomethylarsonic dicysteine (MADG3ϩ), and dimethylarsinic cysteine (DAMG3ϩ) before DMA3ϩ was produced: iAs5ϩ ϩ 2e 3 iAs3ϩ ϩ GSH 3 ATG3ϩ ϩ CH3ϩ 3 MADG3ϩ 3 MMA3ϩ3 MMA5ϩ and MADG3ϩ ϩ CH3ϩ 3 DAMG3ϩ 3 DMA3ϩ 3 DMA5ϩ Both models start from the reduction of iAs5ϩ to iAs3ϩ and do not further describe the function of the enzyme in the reaction. The highly dissociative ability of the enzyme-AdoMet-reductant and the increased content of -pleated sheet help enable the binding of iAs3ϩ to the active sites
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