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.
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- Becker M. A., Tomkins G. M. Pleiotrophy in a cysteine-requiring mutant of Samonella typhimurium resulting from altered protein-protein interaction. J Biol Chem. 1969 Nov 10;244(21):6023–6030. [PubMed] [Google Scholar]
- Cook P. F., Wedding R. T. Cysteine synthetase from Salmonella typhimurium LT-2. Aggregation, kinetic behavior, and effect of modifiers. J Biol Chem. 1978 Nov 10;253(21):7874–7879. [PubMed] [Google Scholar]
- Denk D., Böck A. L-cysteine biosynthesis in Escherichia coli: nucleotide sequence and expression of the serine acetyltransferase (cysE) gene from the wild-type and a cysteine-excreting mutant. J Gen Microbiol. 1987 Mar;133(3):515–525. doi: 10.1099/00221287-133-3-515. [DOI] [PubMed] [Google Scholar]
- Kredich N. M., Becker M. A., Tomkins G. M. Purification and characterization of cysteine synthetase, a bifunctional protein complex, from Salmonella typhimurium. J Biol Chem. 1969 May 10;244(9):2428–2439. [PubMed] [Google Scholar]
- Lunn J. E., Droux M., Martin J., Douce R. Localization of ATP Sulfurylase and O-Acetylserine(thiol)lyase in Spinach Leaves. Plant Physiol. 1990 Nov;94(3):1345–1352. doi: 10.1104/pp.94.3.1345. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rolland N., Droux M., Lebrun M., Douce R. O-acetylserine(thiol)lyase from spinach (Spinacia oleracea L.) leaf: cDNA cloning, characterization, and overexpression in Escherichia coli of the chloroplast isoform. Arch Biochem Biophys. 1993 Jan;300(1):213–222. doi: 10.1006/abbi.1993.1030. [DOI] [PubMed] [Google Scholar]
- Smith I. K. Studies of l-Cysteine Biosynthetic Enzymes in Phaseolus vulgaris L. Plant Physiol. 1972 Oct;50(4):477–479. doi: 10.1104/pp.50.4.477. [DOI] [PMC free article] [PubMed] [Google Scholar]