Purification and characterization of O-acetylserine (thiol) lyase from spinach chloroplasts.
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.
Droux
M
M
UM 41 CNRS, Rhône Poulenc Agrochimie, Centre de la Recherche de la Dargoire, Lyon, France.
Martin
J
J
Sajus
P
P
Douce
R
R
D000123:Acetyltransferases / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D015153:Blotting, Western; D002736:Chloroplasts / Q000201:enzymology*; D002853:Chromatography, Liquid; D003429:Cross Reactions; D003545:Cysteine / Q000096:biosynthesis; D003547:Cysteine Synthase / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes; D008928:Mitochondria / Q000201:enzymology; D010944:Plants / Q000201:enzymology*; D011732:Pyridoxal Phosphate / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D051050:Serine O-Acetyltransferase; D013050:Spectrometry, Fluorescence
D000596:Amino Acids; D000939:Epitopes; D012694:Serine; D011732:Pyridoxal Phosphate; D000123:Acetyltransferases; D051050:Serine O-Acetyltransferase; D003547:Cysteine Synthase; D003545:Cysteine