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B6db references: 0..pkpsaat

type Journal Article
authors Ruffet, M. L.; Droux, M.; Douce, R.
title Purification and Kinetic Properties of Serine Acetyltransferase Free of O-Acetylserine(thiol)lyase from Spinach Chloroplasts
journal Plant Physiol
ui 0; PKPSAAt
year (1994)
volume 104
number 2
pages 597-604.
abstract Serine acetyltransferase, a key enzyme in the L-cysteine biosynthetic pathway, was purified over 300,000-fold from the stroma of spinach (Spinacia oleracea) leaf chloroplasts. The purification procedure consisted of ammonium sulfate precipitation, anion-exchange chromatography (Trisacryl M DEAE and Mono Q HR10/10), hydroxylapatite chromatography, and gel filtration (Superdex 200). The purified enzyme exhibited a specific activity higher than 200 units mg-1 and a subunit molecular mass of about 33 kD upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Moreover, the purified serine acetyltransferase appeared to be essentially free of O- acetyleserine(thiol)lyase, another enzyme component in the L-cysteine biosynthetic pathway. A steady-state kinetic analysis indicated that the mechanism of the enzyme-catalyzed reaction involves a double displacement. The apparent Km for the two substrates, L-serine and acetyl-coenzyme A, were 2.29 [plus or minus] 0.43 and 0.35 [plus or minus] 0.02 mM, respectively. The rate of L-cysteine synthesis in vitro was measured in a coupled enzyme assay using extensively purified O- acetylserine(thiol)lyase and serine acetyltransferase. This rate was maximum when the assay contained approximately a 400-fold excess of O- acetylserine(thiol)lyase over serine acetyltransferase. Measurements of the relative level of O-acetylserine(thiol)lyase and serine acetyltransferase activities in the stroma indicated that the former enzyme was present in much larger quantities than the latter. Thus, the activity ratio for these two enzymes [O-acetylserine(thiol)lyase activity/serine acetyltransferase activity] measured in the stromal protein extract was 345. This strongly suggested that all the O- acetylserine(thiol)lyase and serine acetyltransferase activities in the stroma are involved in bringing a full synthesis of L-cysteine in the chloroplast.
last changed 2003/03/17 14:53

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