Cysteine was used as an alternative thiol reductant to GSH to show that a thiol-arsenical complex, not restricted to As-GSH, is the substrate for arsenite methylation. Our results suggest that the valence state of arsenic is unchanged when the methyl transfer step occurs on h AS3MT.

Reductant reduces the disulfide bond between the cysteine residues of h AS3MT, thereby exposing the active sites for binding to i As, respectively).

A thiol-arsenical complex might also be formed between arsenicals and the active site cysteine residues of h AS3MT.

Significance: This work clearly elucidates the completely ordered mechanism of arsenite methylation by a rapid equilibrium kinetic model.

In the human body, arsenic is metabolized by methylation.

Background: Oxidative methylation and successive methylation are two possible enzymatic mechanisms of arsenite methylation.

Results: Rapid equilibrium kinetic analysis established that h AS3MT-catalyzed arsenite methylation is a completely ordered reaction.

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 absorbance at 412 nm was then monitored to estimate the number of cysteine residues in h AS3MT.The effect of the reductant on the secondary structure was measured by CD spectroscopy after the reduced enzyme was thoroughly dialyzed against phosphate buffer at 4 °C.Arsenicals were analyzed by HPLC-inductively coupled plasma-MS (28).The methylation rates were calculated as mole equivalents of methyl groups transferred from Ado Met to arsenic (, p H 7.0) at 25 °C for 90 min.The relationship between arsenic and its related diseases is complicated by many aspects, such as dose-response relationships, oxidative stress, cellular signaling, cell cycle control, gene amplification, and chromosomal abnormalities (511), both of which have been detected in human urine (12).