Evidence for direct methyl transfer in betaine: homocysteine S-methyl-transferase.

We described recently the purification and preliminary characterization of human hepatic betaine: homocysteins S-methyltransferase (Skiba, W. E., Taylor, M. P., Wells, M. S., Mangum, J. H., and Awad, W. M., Jr. (1982) J. Biol. Chem. 258, 14944-14948) where it was shown that isovalerate and 3,3-dimethylbutyrate, analogs of dimethylglycine and betaine, respectively, were good inhibitors. The present study demonstrates that butyrate is a modest competitive inhibitor, binding at the betaine site. This led to the consideration and synthesis of a putative dual-substrate analog, S(delta-carboxybutyl)-DL-homocysteine, which bound with high affinity to the active site of the methyltransferase; presumably this effect is due to the L-isomer only. Homologs, S(gamma-carboxypropyl)-DL-homocysteine and S(beta-carboxyethyl)-DL-homocysteine, do not inhibit at concentrations 100-fold higher than where inhibition is noted with the dual-substrate analog, indicating the latter's specificity. These findings support the hypothesis that methyl transfer in this enzyme occurs directly from one substrate to the other.

Human hepatic methionine biosynthesis. Purification and characterization of betaine:homocysteine S-methyltransferase.

Human hepatic betaine:homocysteine S-methyltransferase has been purified to apparent homogeneity after a 250-fold separation. The isolation required the presence of homocysteine and a product of the reaction, dimethylglycine, in order to stabilize the protein. An apparent molecular weight of 270,000 was determined by calibrated gel filtration. A single peptide chain of Mr = 45,000 was found by calibrated sodium dodecyl sulfate-acrylamide gel electrophoresis, suggesting that the native protein is a hexamer of identical subunits. The enzyme is stable at pH values greater than 5.5. No effect of EDTA was observed on the activity of the enzyme. In the absence of thiol reagents, the hexameric protein appeared to polymerize to integral aggregates. Isovalerate and 3,3-dimethylbutyrate, analogs of dimethylglycine and betaine, respectively, are good inhibitors of the enzyme. The inhibitions are competitive with respect to betaine, indicating that a positive charge is not required for binding at the betaine/dimethylglycine site. These findings are similar to those reported for acetylcholine esterase where the neutral analogs of choline show good binding to that enzyme. The binding site for betaine/dimethylglycine may exist in two states, one permitting the binding of a positively charged group and the other a neutral group.