Methylation of Inorganic Arsenic and Mechanism of the Human Arsenic (+3Oxidation State)Methyltransferase
|Keywords||Inorganic arsenic Arsenic (+3oxidation state) methyltransferase Methylation of inorganic arsenic Cysteine residues Structure-function roles Thermotropic properties Enzyme mechanism Transition metal ions Selenite Inhibition mechanism|
The methylation of inorganic arsenic (iAs) is the main metabolism pathway in mammals and has been commonly considered as a detoxification mechanism. In this dissertation, we studied the methylation of iAsⅢ with the purified recombinant human arsenic (+3oxidation state) methyltransferase in vitro. Enzyme kinetics, initial velocity, spectroscopy and HPLC-ICP-MS were used to systematically study the roles of cysteine (Cys) residues in the hAS3MT; to probe the mechanism of the hAS3MT; in addition, the inhibition of the iAsⅢ methylation with the hAS3MT by selenite/transition metal ions were also studied and the inhibition mechanism was proposed and discussed. The main works of this dissertation are as follows:(1) Site-directed mutagenesis of the hAS3MT was performed directly on the cDNA encoding for the hAS3MT in the plasmid pET-32a-hAS3MT. Substitutions of serine for Cys72, Cys271, Cys334, Cys360and Cys375of the hAS3MT were carried out using the PCR megaprimer or PCR overlap extension method with pET-32a-hAS3MT as the DNA template. DNA sequencing of the hAS3MT mutants was carried out using the double-stranded dideoxy method to ensure that no errors had been introduced during the amplification processes. E. coli BL21(DE3) pLysS was used for protein production and the target proteins were purified by Ni-NTA agarose column. The mutants were systematically assayed with respect to enzymatic activity, kinetics, thermal stability, secondary structures as well as inhibition by selenite (Se). Present results indicate that C72S is completely inactive in the methylation of iAsⅢ and has distinct changes in the secondary structures. Cys72might form a critical intramolecular disulfide bond with Cys250. Cys271and Cys375do not affect the activity and structure of the hAS3MT. However, Cys334and Cys360have some effects on the function of the hAS3MT, the mutation of these two residues decreased the enzymatic turnovers. The kinetic data indicate that Cys271, Cys334, Cys360and Cys375are not involved in the S-adenosylmethionine (SAM) binding but can bind iAsⅣ and SeⅣ. In addition, all these Cys residues except Cys375could affect the thermotropic properties of the hAS3MT.(2) Soluble and active hAS3MT in the E. coli BL21(DE3) pLysS was expressed under a lower induction temperature of25℃and the target protein was purified by Ni-NTA agarose column. The catalytic mechanism of the recombinant hAS3MT was studied using kinetics, initial velocity and spectroscopy. The production and the distribution of methylated arsenicals changed with various concentrations of iAsⅢ/SAM/thiols, enzyme contents and incubation times. These results suggest a sequential methylation of iAsⅢ to monomethylated arsenicals (MMA) and dimethylated arsenicals (DMA); also competition exists between the two methylation reactions. hAS3MT showed its greatest activity at pH8.5with glutathione (GSH) as the reductant. Cys156and Cys206have been proved to be the active sites of the hAS3MT. The pKa of sulfhydryl group in Cys residue is about8.3. The optimal weak alkaline environment (pH8.5) can facilitate the balance between the deprotonation and the protonation of these two active sites to expedite the reaction. Detailed initial velocity studies were carried out by varying the concentration of one reactant in the presence of different fixed concentrations of the second reactant, while the concentrations of the other reactants were held constant. The results illuminate an ordered sequence for the binding of SAM and iAsⅢ to hAS3MT. Differently, GSH should probably be placed either as the first reactant or as a reactant combining with the enzyme only after one or more products have been released. The interactions between substrate/cofactors and the hAS3MT were first monitored by UV-visible, and circular dichroism (CD) spectroscopy. It reveals that iAsⅢ and SAM combine with the hAS3MT before reaction starts;whereas, no interactions between GSH and the hAS3MT are detected by UV-vis and CD spectra. Based on all the results and discussions above, a mechanism (Scheme1) which is essentially consistent with Hayawaba’s proposal is proposed. However, As-thiol complex is not a requirement for iAsⅢ as the substrate in our scheme. The hAS3MT first combines with SAM, and then iAsⅢ adds to it, forming a ternary complex. After that, iAsⅢ is successively methylated reductively, rather than a stepwise oxidative methylation. A disulfide bond forms in the enzyme at the end of the catalytic cycle. Then reductant (thiols or non-sulfhydryl reductants that can reduce disulfide bond) reacts with the disulfide bond to resume the active form of the enzyme.(3) Since iAs can cause liver cancer, hemolysis, marrow suppression, and blood vessels thickening, we chose the transition metals (CoCl2, ZnCl2, MnCl2,FeCl2) which respectively show high concentration in the liver and marrow, skeletal muscle, bones, and blood to study their influence on the methylation of iAsⅢ by the hAS3MT. As well, more studies were considered to probe the inhibition mechanism of SeⅣ and the metal ions. UV-visible, CD and fluorescence spectroscopy were used to study the interactions between the metal ions above and the hAS3MT. The results showed that CoⅡ, MnⅡ and Zn" inhibited the iAsⅢ methylation with hAS3MT in a concentration-dependent manner and the kinetics data indicated that CoⅡ and MnⅡ were similar with mixed (competitive and non-competitive) inhibitors while ZnⅡ was a competitive inhibitor. However, only a high concentration of FeⅡ could restrain the methylation. Further studies showed that neither superoxide anion nor hydrogen peroxide was involved in the inhibition of transition metal ion or SeⅣ for the hAS3MT activity. The inhibition of iAsⅢ methylating activity of the hAS3MT by SeⅣ was reversed by2mM DTT (dithiothreitol) but neither by cystenie (CySH) nor by P-mercaptoethanol (ME). Whereas, besides DTT, CySH can also prevent the inhibition of hAS3MT activity by CoⅡ, MnⅡ and ZnⅡ. The results from DTNB assays showed that free Cys residues were involved in the interactions of transition metal ions or SeⅣ with the hAS3MT. It is proposed that the inhibitory effect of the ions (CoⅡ, MnⅡ and ZnⅡ) or SeⅣ on the hAS3MT activity might be via the interactions of them with amino acid residues (including Cys residues) in the hAS3MT to form inactive protein adducts. The inhibitory effect of SeⅣ and ZnⅡ on the hAS3MT activity was almost via a RS-Zn/Se-SR type. For the inhibition by CoⅡ and MnⅡ, in addition to the RS-Co/Mn-SR type, disulfide type perhaps was also used.