Rapid purification and characterization of homoserine dehydrogenase from Saccharomyces cerevisiae.
Homoserine dehydrogenase of Saccharomyces cerevisiae has been rapidly purified to homogeneity by heat and acid treatments, ammonium sulfate fractionation, and chromatography on Matrex Gel Red A and Q-Sepharose columns. The final preparation migrated as a single entity upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a Mr of 40,000. The Mr of the native enzyme was 81,000 as determined by gel filtration, suggesting that the enzyme is composed of two identical subunits. This feature was also confirmed by cross-linking analysis using the bifunctional reagent dimethyl suberimidate. Feedback inhibition by L-methionine and L-threonine was observed using the purified enzyme. The enzyme was markedly stabilized against heat treatment at high salt concentrations. Additions of feedback inhibitors or high concentrations of salts failed to cause any dissociation or aggregation of the enzyme subunits unlike enzymes from other sources such as Rhodospirillum rubrum. The enzyme denatured in 3 M guanidine-HCl was refolded by simple dilution with a concomitant restoration of the activity. Cross-linking analysis of the renaturation process suggested that the formation of the dimer is required for activity expression. Amino acid sequence analysis of peptides obtained by digestion of the enzyme protein with Achromobacter lyticus protease I revealed that several amino acid residues are strictly conserved among homoserine dehydrogenases from S. cerevisiae, Escherichia coli, and Bacillus subtilis.
Yumoto
N
N
Department of Chemistry, Faculty of Science, Kyoto University, Japan.
Kawata
Y
Y
Noda
S
S
Tokushige
M
M
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000645:Ammonium Sulfate; D002845:Chromatography; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005590:Fractional Precipitation; D006715:Homoserine Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011489:Protein Denaturation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012689:Sequence Homology, Nucleic Acid
D000596:Amino Acids; D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D006715:Homoserine Dehydrogenase; D000645:Ammonium Sulfate