Droux M, Martin J, Sajus P, Douce R
UM 41 CNRS, Rhône Poulenc Agrochimie, Centre de la Recherche de la Dargoire, Lyon, France.
Arch Biochem Biophys. 1992 Jun;295(2):379-90. doi: 10.1016/0003-9861(92)90531-z.
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.
O-乙酰丝氨酸（硫醇）裂解酶是半胱氨酸生物合成途径中的最后一种酶，已从菠菜叶叶绿体中纯化至同质。该酶的分子量为68,000，由两个分子量为35,000的相同亚基组成。在pH 7.5下获得的吸收光谱由于磷酸吡哆醛在407 nm处有一个峰值，添加O-乙酰丝氨酸会导致光谱发生相当大的变化。发现磷酸吡哆醛含量为每35,000亚基1.1个，发色团被O-乙酰丝氨酸从酶上取代，导致全酶逐渐失活。在Superdex 200上进行凝胶过滤色谱时，部分叶绿体O-乙酰丝氨酸（硫醇）裂解酶与丝氨酸乙酰转移酶一起在对应于分子量310,000的位置洗脱（这种称为半胱氨酸合酶的复合物已在细菌中得到表征）。O-乙酰丝氨酸（硫醇）裂解酶的活性在pH 7.5至8.5之间最佳。O-乙酰丝氨酸的表观Km为1.3 mM，硫化物的表观Km为0.25 mM。在10 mM O-乙酰丝氨酸时计算出的活化能为12.6 kcal/mol。菠菜叶绿体O-乙酰丝氨酸（硫醇）裂解酶的整体氨基酸组成与从菠菜叶粗提物中获得的相同酶（胞质的？）的氨基酸组成不同。针对叶绿体O-乙酰丝氨酸（硫醇）裂解酶制备的多克隆抗体与线粒体基质和胞质蛋白制剂的交叉反应性非常低，这表明叶绿体同工型与线粒体和胞质对应物不同。
