O-Acetylhomoserine sulfhydrylase of the fission yeast Schizosaccharomyces pombe: partial purification, characterization, and its probable role in homocysteine biosynthesis.
A crude extract of Schizosaccharomyces pombe cells catalyzed sulfhydrylation of both O-acetyl-L-serine and O-acetyl-L-homoserine with H2S, but did not synthesize cystathionine from O-acetyl-L-homoserine and L-cysteine. The O-acetylhomoserine sulfhydrylase [EC 4.2.99.10] was very unstable; however, it could be stabilized by the addition of 25% (w/w) sucrose or glycerol. The optimal pH for activity was 8.0 and that for stability was 7.0. The enzyme was purified approximately 300-fold from an ammonium sulfate-precipitated fraction. L-Methionine was the most effective inhibitor among the amino acids examined. It inhibited the enzyme competitively with respect to OAH with a Ki value of 2.6 mM. Sulfhydrylase activity was inhibited to various extents by some carbonyl reagents, but sulfhydryl reagents such as p-chloromercuribenzoic acid, 5,5'-dithio-bis(2-nitrobenzoic acid), and monoiodoacetic acid had no inhibitory effect. The enzyme also reacted with O-succinylhomoserine and L-homoserine to synthesize homocysteine directly, but could not utilize cysteine as a co-substrate in place of H2S. In the sulfhydrylation reactions, Km values for the substrates ranged from 10.4-12.5 mM. The enzyme was resolved to the apoenzyme by incubation with phenylhydrazine and reactivated by the addition of pyridoxal 5'-phosphate, whose Km value was 0.083 microM. The molecular weight of the enzyme was estimated to be approximately 186,000 by gel filtration and 170,000 by ultracentrifugation in sucrose density gradients. The isolectric point of the protein was pH 4.1. The characteristics of this enzyme are compared with those of physiologically functional sulfhydrylases reported for other organisms, and the possibility of the enzyme functioning as a homocysteine synthase is discussed.
D000603:Amino Acids, Sulfur / Q000096:biosynthesis; D001203:Ascomycota / Q000201:enzymology*; D019757:Carbon-Oxygen Lyases; D002384:Catalysis; D055598:Chemical Phenomena; D002621:Chemistry; D002850:Chromatography, Gel; D002855:Chromatography, Thin Layer; D003080:Cold Temperature; D003547:Cysteine Synthase; D004589:Electrophoresis, Disc; D006710:Homocysteine / Q000096:biosynthesis*; D007526:Isoelectric Point; D007700:Kinetics; D008190:Lyases / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D009097:Multienzyme Complexes; D011489:Protein Denaturation; D029701:Saccharomyces cerevisiae Proteins; D012568:Schizosaccharomyces / Q000201:enzymology*
D000603:Amino Acids, Sulfur; D009097:Multienzyme Complexes; D029701:Saccharomyces cerevisiae Proteins; D006710:Homocysteine; D003547:Cysteine Synthase; C103381:MET17 protein, S cerevisiae; C018725:O-acetylhomoserine (thiol)-lyase; D008190:Lyases; D019757:Carbon-Oxygen Lyases