O-Acetylserine and O-acetylhomoserine sulfhydrylase of yeast. Subunit structure.
The molecular weight of O-acetylserine (OAS)-O-acetylhomoserine (OAH) sulfhydrylase purified from yeast was estimated to be about 200,000 by Sephadex G-200 gel chromatography in various buffers. The S20, w value of this protein was determined to be about 9.0 by sucrose density gradient centrifugation. The calculated molecular weight based on this value was similar to that estimated by gel chromatography. Treatment with 1% sodium dodesylsulfate (SDS) or 6 M urea dissociated the enzyme into 4 subunits; these had a molecular weight estimated to be 51,000 by SDS-poly-acrylamide gel electrophoresis and to be 57,000 by Sephadex G-100 gel chromatography in the presence of 6 M urea and 0.5% beta-mercaptoethanol. The 4 subunits appeared to be identical, based on the symmetric subunit elution pattern from a Sephadex column, a single peptide band on SDS-polyacrylamide gel, and the detection of histidine as the sole N-terminal amino acid in the native enzyme. Since dissociation into the subunits occurred without the use of reducing agents, the association of the subunits seems to require no disulfide linkage. One mole of the subunit contained one mole of sulfhydryl group which appeared to be buried inside the molecule. Partial restoration of the catalytic activity was observed when the urea-denatured enzyme was dialyzed to remove urea, especially in the presence of reducing agents such as dithiothreitol. The urea-denatured enzyme showed a tendency in the absence of reducing agents to form a subunit dimer linked by a disulfide bond between the cystine residues exposed by denaturation. The amino acid composition of the enzyme was determined; it contained one half-cystine residue per subunit, and the content of acidic residues was much higher than that of basic residues. Based on these findings, the subunit structure of the enzyme is discussed.
D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D003547:Cysteine Synthase; D006714:Homoserine / Q000031:analogs & derivatives; D008190:Lyases; D046911:Macromolecular Substances; D008970:Molecular Weight; D009097:Multienzyme Complexes; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013438:Sulfhydryl Compounds / Q000032:analysis; D013439:Sulfhydryl Reagents
D000596:Amino Acids; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D006714:Homoserine; D003547:Cysteine Synthase; D008190:Lyases