Purification and kinetic properties of serine acetyltransferase free of O-acetylserine(thiol)lyase from spinach chloroplasts

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...

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Bibliographic Details
Published in:Plant physiology (Bethesda) 1994-02, Vol.104 (2), p.597-604
Main Authors: Ruffet, M.L, Droux, M, Douce, R
Format: Article
Language:English
Subjects:
ACILTRANSFERASA
ACTIVIDAD ENZIMATICA
ACTIVITE ENZYMATIQUE
ACYLTRANSFERASE
Biological and medical sciences
BIOSINTESIS
BIOSYNTHESE
CHLOROPLASTE
Chloroplasts
Chromatography
CISTEINA
CLOROPLASTO
COMPOSICION QUIMICA
COMPOSITION CHIMIQUE
CYSTEINE
Enzymes
FEUILLE
Fundamental and applied biological sciences. Psychology
Gels
HOJAS
Kinetics
LIASAS
Life Sciences
LYASE
Metabolism
Metabolism and Enzymology
Plant physiology and development
Plants
PURIFICACION
PURIFICATION
SERINA
SERINE
Sodium
Spinach
SPINACIA OLERACEA
Sulfides
Weddings
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Summary: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-acetylserine(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 +/- 0.43 and 0.35 +/- 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
ISSN:0032-0889
1532-2548
DOI:10.1104/pp.104.2.597