Purification and characterization of O-acetylserine (thiol) lyase from spinach chloroplasts

Arch Biochem Biophys. 1992 Jun;295(2):379-90. doi: 10.1016/0003-9861(92)90531-z.

Abstract

O-Acetylserine (thiol) lyase, the last enzyme in the cysteine biosynthetic pathway, was purified to homogeneity from spinach leaf chloroplasts. The enzyme has a molecular mass of 68,000 and consists of two identical subunits of Mr 35,000. The absorption spectrum obtained at pH 7.5 exhibited a peak at 407 nm due to pyridoxal phosphate, and addition of O-acetylserine induced a considerable modification of the spectrum. The pyridoxal phosphate content was found to be 1.1 per subunit of 35,000, and the chromophore was displaced from the enzyme by O-acetylserine, leading to a progressive inactivation of the holoenzyme. Upon gel filtration chromatography on Superdex 200, part of the chloroplastic O-acetylserine (thiol) lyase eluted in association with serine acetyltransferase at a position corresponding to a molecular mass of 310,000 (such a complex called cysteine synthase has been characterized in bacteria). The activity of O-acetylserine (thiol) lyase was optimum between pH 7.5 and 8.5. The apparent Km for O-acetylserine was 1.3 mM and for sulfide was 0.25 mM. The calculated activation energy was 12.6 kcal/mol at 10 mM O-acetylserine. The overall amino-acid composition of spinach chloroplast O-acetylserine (thiol) lyase was different than that determined for the same enzyme (cytosolic?) obtained from a crude extract of spinach leaves. A polyclonal antibody prepared against the chloroplastic O-acetylserine (thiol) lyase exhibited a very low cross-reactivity with a preparation of mitochondrial matrix and cytosolic proteins suggesting that the chloroplastic isoform was distinct from the mitochondrial and cytosolic counterparts.

MeSH terms

  • Acetyltransferases / metabolism
  • Amino Acids / analysis
  • Blotting, Western
  • Chloroplasts / enzymology*
  • Chromatography, Liquid
  • Cross Reactions
  • Cysteine / biosynthesis
  • Cysteine Synthase / chemistry
  • Cysteine Synthase / immunology
  • Cysteine Synthase / isolation & purification*
  • Cysteine Synthase / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Epitopes
  • Mitochondria / enzymology
  • Plants / enzymology*
  • Pyridoxal Phosphate / metabolism
  • Serine / metabolism
  • Serine O-Acetyltransferase
  • Spectrometry, Fluorescence

Substances

  • Amino Acids
  • Epitopes
  • Serine
  • Pyridoxal Phosphate
  • Acetyltransferases
  • Serine O-Acetyltransferase
  • Cysteine Synthase
  • Cysteine