| BHMT is found in the liver and kidney of mammals and catalyzes the transfer of a methyl group from betaine to homocysteine, producing dimethylglycine and methionine. The sulfonium analog of betaine, dimethylacetothitin (DMAT) is an efficient methyl donor in the BHMT reaction. We have discovered that BHMT is a Zn metalloenzyme, and that Zn is required for catalysis.; We have found that BHMT can also methylate Asp when DMAT is used as methyl donor. This assay made it possible to show that BHMT has an absolute requirement for a reducing agent. Enzyme buffer devoid of reducing agent is inactive, and activity is restored upon addition of reducing agent. BHMT contains eight cysteine residues; three (Cys217, Cys299, and Cys300) have been found to donate sulfur ligands to catalytic Zn. Treatment with dithionitrobenzoate (DTNB) suggests seven are reactive. A BHMT mutant (5Cys/Ala) was made that had all five Cys residues not involved in Zn-binding changed to Ala. 5Cys/Ala retained normal activity in the betaine-Hcy or the reducing agent-dependent DMAT-Asp assays. Thus, the Cys residues that are not ligands to Zn are not essential for catalysis and do have a role in the redox phenomena.; The lack of activity in the absence of reducing agent is not due to loss of Zn at the catalytic site. When reducing agent-free preparations of 5Cys/Ala are incubated with the Zn chelator, PAR, and the absorbence of the Zn-PAR chelate is monitored, there is no change in absorbence unless methyl-methanethiosulfonate or H2O2 are added, and then the amount of Zn released from the enzyme is equivalent to the amount of 5Cys/Ala in solution. Finally, when BHMT-5Cys/Ala is in the presence or absence of reducing agent, and in the former condition the reducing agent is removed by gel filtration, the number of DTNB-modifiable residues is three and one, respectively. These data indicate that the redox effect on BHMT activity is mediated by the formation of a reversible disulfide bond between two of the three thiolates that normally chelate Zn. When oxidized, the loss of activity is likely due to the change in the electronics of the Zn-ligand interactions. |
