Acetyl-CoA pathway of autotrophic growth. Identification of the methyl-binding site of the CO dehydrogenase.

Abstract

CO dehydrogenase, a key enzyme of the acetyl-CoA pathway of autotrophic growth, has been methylated using 14CH3I or 14CH3-corrinoid enzyme plus ferredoxin. Acetyl-CoA was synthesized from the resulting 14CH3-CO dehydrogenase, CO, and CoASH, with about 50% yield of the available 14C and without addition of other enzymes except CO dehydrogenase disulfide reductase. Even the reductase could be replaced by dithioerythritol. Amino acid analysis of the 14CH3-CO dehydrogenase showed two radioactive peaks, one of which migrated as S-methylcysteine but very close to the methyl ester of glutamic acid. By oxidation with H2O2, the radioactive component of this peak was identified as S-methylcysteine sulfone. Amino acid analysis of the 14CH3-CO dehydrogenase after synthesis of acetyl-CoA demonstrated that there was a large decrease in radioactivity of the peak containing the S-methyl-cysteine. The compound present in the second peak has not been identified; there was no decrease in its radioactivity. By nonreducing gel electrophoresis of the 14CH3-CO dehydrogenase, followed by autoradiography, it was shown that the beta subunit is the methyl acceptor. These results demonstrate that a cysteine of the beta subunit is the methyl acceptor and that CO dehydrogenase per se catalyzes the synthesis of acetyl-CoA.