Homocysteine Synthesis in Yeast: Partial Purification and Properties of O-Acetylhomoserine Sulfhydrylase*

O-Acetylhomoserine sulfhydrylase, catalyzing the synthesis of homocysteine from O-acetylhomoserine and hydrogen sulfide, was purified more than 100-fold from baker's yeast mainly by means of DEAE-cellulose column chromatography. Polyacrylamide gel electrophoresis revealed that the purified preparation consisted of at least three protein components, of which only the main component possessed the sulfhydrylase activity. As revealed with Neurospora enzyme, this enzyme also required O-acetyl-L-homoserine as a substrate, but pyridoxal 5-phosphate was required for its activity. The Km values of the enzyme for O-acetyl-L-homoserine and pyridoxal 5-phosphate were determined to be 4.55×10−3M and 2.94×10−5M, respectively. Hydrogen sulfide could be replaced by methyl and ethyl mercaptans giving rise to methionine and ethionine, respectively. Cystathionine was not synthesized from O-acetylhomoserine and cysteine in the presence of the enzyme. O-Acetyl-L-serine (K1= 21.8mM), O-succinyl-DL-homoserine (K1 = 19.0 mM), L-homoserine (K1=16.0mM) and L-methionine inhibited the enzyme in competition with O-acetylhomoserine.