Partial purification and some properties of homoserine O-acetyltransferase of a methionine auxotroph of Saccharomyces cerevisiae.
A wild-type strain and six methionine auxotrophs of Saccharomyces cerevisiae were cultured in a synthetic medium supplemented with 0.1 mM L-cysteine or L-methionine and analyzed for the synthesis of homoserine O-acetyltransferase (EC 2.3.1.31). Among them, four mutant strains exhibited enzyme activity in cell extracts. Methionine added to the synthetic medium at concentrations higher than 0.1 mM repressed enzyme synthesis in two of these strains. The enzyme was partially purified (3,500-fold) from an extract of a mutant strain through ammonium sulfate fractionation and chromatography on columns of DEAE-cellulose, Phenyl-Sepharose C1-4B, and Sephadex G-150. The enzyme exhibited optimal pH at 7.5 for activity and at 7.8 for stability. The reaction product was ascertained to be O-acetyl-L-homoserine by confirming that it produced L-homocysteine in an O-acetyl-L-homoserine sulfhydrylase reaction. The Km for L-homoserine was 1.0 mM, and for acetyl coenzyme A it was 0.027 mM. The molecular weight of the enzyme was estimated to be approximately 104,000 by Sephadex G-150 column chromatography and 101,000 by sucrose density gradient centrifugation. The isoelectric point was at pH 4.0. Of the hydroxy amino acids examined, the enzyme showed reactivity only to L-homoserine. Succinyl coenzyme A was not an acyl donor. In the absence of L-homoserine, acetyl coenzyme A was deacylated by the enzyme, with a Km of 0.012 mM. S-Adenosylmethionine and S-adenosylhomocysteine slightly inhibited the enzyme, but methionine had no effect.
Yamagata
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D000123:Acetyltransferases / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism; D004794:Enzyme Repression; D006714:Homoserine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008715:Methionine / Q000378:metabolism* / Q000494:pharmacology; D008970:Molecular Weight; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000378:metabolism; D013379:Substrate Specificity
D006714:Homoserine; D008715:Methionine; D000123:Acetyltransferases; C021369:homoserine O-acetyltransferase