Identification of the Third Binding Site of Arsenic in Human Arsenic (III) Methyltransferase

Abstract

Arsenic (III) methyltransferase (AS3MT) catalyzes the process of arsenic methylation. Each arsenite (iAs3+) binds to three cysteine residues, methylarsenite (MMA3+) binds to two, and dimethylarsenite (DMA3+) binds to one. However, only two As-binding sites (Cys156 and Cys206) have been confirmed on human AS3MT (hAS3MT). The third As-binding site is still undefined. Residue Cys72 in Cyanidioschyzon merolae arsenite S-adenosylmethyltransferase (CmArsM) may be the third As-binding site. The corresponding residue in hAS3MT is Cys61. Functions of Cys32, Cys61, and Cys85 in hAS3MT are unclear though Cys32, Cys61, and Cys85 in rat AS3MT have no effect on the enzyme activity. This is why the functions of Cys32, Cys61, and Cys85 in hAS3MT merit investigation. Here, three mutants were designed, C32S, C61S, and C85S. Their catalytic activities and conformations were determined, and the catalytic capacities of C156S and C206S were studied. Unlike C85S, mutants C32S and C61S were completely inactive in the methylation of iAs3+ and active in the methylation of MMA3+. The catalytic activity of C85S was also less pronounced than that of WT-hAS3MT. All these findings suggest that Cys32 and Cys61 markedly influence the catalytic activity of hAS3MT. Cys32 and Cys61 are necessary to the first step of methylation but not to the second. Cys156 and Cys206 are required for both the first and second steps of methylation. The SC32 is located far from arsenic in the WT-hAS3MT-SAM-As model. The distances between SC61 and arsenic in WT-hAS3MT-As and WT-hAS3MT-SAM-As models are 7.5 Å and 4.1 Å, respectively. This indicates that SAM-binding to hAS3MT shortens the distance between SC61 and arsenic and promotes As-binding to hAS3MT. This is consistent with the fact that SAM is the first substrate to bind to hAS3MT and iAs is the second. Model of WT-hAS3MT-SAM-As and the experimental results indicate that Cys61 is the third As-binding site.

Determinants and Consequences of Arsenic Metabolism Efficiency among 4,794 Individuals: Demographics, Lifestyle, Genetics, and Toxicity

“…DMA loaded negatively (−100) on PC1 (eigenvalue = 2.08), and MMA (67) and iAs (80) both loaded positively. For PC2 (eigenvalue = 0.93), MMA loaded positively (75), iAs loaded negatively (−60), and DMA had a loading near zero (2). We multiplied PC1 by −1 so that higher PCs scores represent more methylation (i.e, more DMA% and MMA% respectively).…”

“…However, the mechanism(s) by which these SNPs influence metabolism remain unclear. Kinetic studies have shown that the AS3MT binding affinity differs for the first methylation step as compared to the second step (73,74) and there are differences in the number of binding sites required for each of the methylation steps (75). These differences in AS3MT kinetics for the first vs. the second methylation step suggest that it possible that the SNPs in this region could have different effects on these two methylation reactions this population.…”

The Functions of Crucial Cysteine Residues in the Arsenite Methylation Catalyzed by Recombinant Human Arsenic (III) Methyltransferase

“…Thomas et al and our group have analyzed the functions of these crucial Cys residues and have confirmed that Cys61, Cys156, and Cys206 are the active sites of hAS3MT [26] – [30] . Like Cys72 in CmArsM, Cys61 moves toward Cys156 and Cys206 upon AdoMet binding, and leaves away after the first step methylation [30] . However, it is not clear if a disulfide bond is formed between the active residues of during the catalytic cycle.…”

“…Previous studies have suggested that Cys72 may be important for the maintenance of hAS3MT conformation [29] , and Cys61 may be the third binding site for iAs 3+ [30] . However, we did not detect the peptides corresponding to Cys61 (IA-modified) and Cys72 (IA-modified) in hAS3MT or its reduced form.…”

“…However, we did not detect the peptides corresponding to Cys61 (IA-modified) and Cys72 (IA-modified) in hAS3MT or its reduced form. Combined with the hAS3MT structural model, based on the crystal structure of CmArsM, we hypothesized that Cys61 and Cys72 might be buried intemally, the in gel IA-modification and trypsin digestion may not be complete [25] , [30] .…”