Cystathionine γ-synthase from Arabidopsis thaliana:: purification and biochemical characterization of the recombinant enzyme overexpressed in Escherichia coli
Cystathionine γ-synthase from Arabidopsis thaliana:: purification and biochemical characterization of the recombinant enzyme overexpressed in Escherichia coli
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DOI：
10.1042/bj3310639
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发表时间：
1998-04-15
影响因子：
4.1
通讯作者：
Douce, R
中科院分区：
文献类型：
--
作者：
Ravanel, S;Gakière, B;Douce, R
胱硫醚γ-合酶催化植物中蛋氨酸生物合成的第一个特异性反应，即用半胱氨酸对O-磷酸高丝氨酸的磷酰基取代基进行γ-取代。编码来自拟南芥的胱硫醚γ-合酶的cDNA已被克隆并用于在大肠杆菌中过表达该酶。天然重组酶是由 53 kDa 亚基组成的同源四聚体，每个亚基与一个在蛋白质前体的赖氨酸 379 处结合的吡哆醛 5'-磷酸分子紧密相关。置换反应遵循 Ping Pong 机制，O-磷酸高丝氨酸和半胱氨酸的 V-max 为 33.6 单位/mg，K-m 值分别为 2.5 mM 和 460 muM。 O-磷酸高丝氨酸对炔丙基甘氨酸引起的酶失活的保护作用表明底物的 K-d 约为。其 K-d 值的 1/2500。因此，大多数这些生化特性与之前报道的植物和细菌胱硫醚γ-合酶的生化特性相似。然而，植物酶与其肠杆菌对应物明显不同，因为它在没有半胱氨酸的情况下催化非常微弱的 O-磷酸高丝氨酸的 γ 消除，该过程大约是 γ 替换反应的 1/2700，大约是 1/2700。速度是大肠杆菌酶催化的 γ 消除速度的 1/1500。这种巨大的差异可能是由于拟南芥胱硫醚γ-合酶无法积累长波长吸收物质，而长波长吸收物质是肠杆菌酶催化的有效γ-消除反应的特征。
Cystathionine gamma-synthase catalyses the first reaction specific for methionine biosynthesis in plants, the gamma-replacement of the phosphoryl substituent of O-phosphohomoserine by cysteine, A cDNA encoding cystathionine gamma-synthase from Arabidopsis thaliana has been cloned and used to overexpress the enzyme in Escherichia coli. The native recombinant enzyme is a homotetramer composed of 53 kDa subunits, each being tightly associated with one molecule of pyridoxal 5'-phosphate that binds at lysine-379 of the protein precursor. The replacement reaction follows a Ping Pong mechanism with a V-max of 33.6 units/mg and K-m values of 2.5 mM and 460 mu M for O-phosphohomoserine and cysteine respectively. The protective effect of O-phosphohomoserine against enzyme inactivation by propargylglycine indicated that the K-d for the substrate is approx. 1/2500 of its K-d value. Thus most of these biochemical properties are similar to those previously reported for plant and bacterial cystathionine gamma-synthases. However, the plant enzyme differs markedly from its enterobacterial counterparts because it catalyses a very faint gamma-elimination of O-phosphohomoserine in the absence of cysteine, this process being about 1/2700 as fast as the gamma-replacement reaction and approx. 1/1500 as fast as the gamma-elimination catalysed by the E. coli enzyme. This huge difference could be attributed to the inability of the A. thaliana cystathionine gamma-synthase to accumulate a long-wavelength-absorbing species that is characteristic for the efficient gamma-elimination reaction catalysed by the enterobacterial enzyme.