A NOVEL PATHWAY FOR THE CONVERSION OF HOMOCYSTEINE TO METHIONINE IN EUKARYOTES

Activation of amino acid homocysteine was compared with that of methio nine in rabbit crude liver extracts and purified multienzyme complex o f aminoacyl-tRNA. synthetases. Activation was studied by measuring the incorporation of radioactive amino acid into unlabelled trichloroacet ic-acid insoluble materials in the absence of protein synthesis. Homoc ysteine synthetase activity was found in the crude extract and in the purified multienzyme complex of aminoacyl-tRNA synthetases. On a molar basis, the activation of methionine by the crude extract was five tim es higher than the activation of homocysteine. There was a partial los s of Hcy-tRNA synthetase activity in the purified multi-enzyme complex . Preliminary reconstitution experiments indicated a requirement for a n additional factor for Hcy-tRNA synthetase activity. TLC of the amino acid released from tRNA charged with [C-14]homocysteine, revealed rad ioactivity in homocysteine, methionine and homocysteine thiolactone, i ndicating a conversion of tRNA-attached homocysteine to methionine. To tal tRNA was separated on a benzoylated cellulose column into a fracti on enriched in initiator tRNA and a methionine-accepting, but initiato r tRNA-deficient, fraction. Homocysteine-accepting activity was presen t only in the initiator tRNA-enriched fraction. Based on the above dat a we propose that homocysteine activation in reticuloqte lysates, repo rted previously, also occurs in liver. Activated homocysteine is attac hed to initiator tRNA and then converted to methionine by a methylatin g enzyme. In the absence of methylation, tRNA-attached homocysteine is hydrolysed to produce homocysteine thiolactone.