Abstract:

S-Adenosyl-L-methionine(AdoMet)-dependent methyltransferases are a widespread class of enzymes exist in both prokaryotes and eukaryotes. Here we report a coupled enzymatic analysis method established to quantitatively characterize S-adenosyl-L-homocysteine(AdoHcy), the common product of AdoMet-dependent methyltransferases. Recombinant S-adenosylhomocysteine nucleosidase(SAHN) and recombinant S-ribosylhomocysteinase(SRHH), which are involved in the enzymatic method, were cloned and expressed as His-tag proteins, and further purified with Ni²⁺ charged agarose resin column, respectively. In this assay, AdoHcy was first hydrolyzed by SAHN into adenine and S-ribosylhomocysteine. Subsequently, the compound S-ribosylhomocysteine generated from SAHN was further cleaved by SRHH to form homocysteine. Finally, homocysteine was quantified using DTNB(Ellman's reagent). The experimental result demonstrated a fine linear positive correlation between formed TNB and initial concentration of AdoHcy, and repeatability was very good. The advantage of this methyltransferase analysis to traditional radioactivity assay is the removal of AdoHcy product by SAHN, which alleviates product feedback inhibition of AdoMet-dependent methyltransferases. This method may be used to analyze other enzymes that produce AdoHcy.

Key words: S-Adenosyl-L-homocysteine(AdoHcy), S-Adenosylhomocysteine nucleosidase(SAHN), S-Ribosylhomocysteinase(SRHH), AdoMet-dependent methyltransferase, Coupled enzymatic analysis