Ravanel S, Droux M, Douce R
Unité Mixte CNRS/Rhône-Poulenc Agrochimie, Centre de Recherche de la Dargoire, Lyon, France.
Arch Biochem Biophys. 1995 Jan 10;316(1):572-84. doi: 10.1006/abbi.1995.1077.
Cystathionine gamma-synthase, the first enzyme specific for the methionine biosynthetic pathway, was purified to apparent homogeneity from spinach leaf chloroplasts. A nonradioactive assay based on O-phthaldialdehyde derivatization of L-cystathionine and fluorescence detection was developed to determine the cystathionine gamma-synthase activity. A unique cystathionine gamma-synthase activity was located in the stromal fraction of chloroplasts while cystathionine beta-lyase, the second enzyme of the transsulfuration pathway, was associated with both the chloroplastic and cytosolic compartments (see companion manuscript). The purified enzyme exhibited a specific activity of 13 U mg-1. As estimated by gel filtration and polyacrylamide gel electrophoresis (PAGE) under nondenaturing conditions followed by activity staining, the native enzyme had an apparent M(r) of 215,000. On the basis of sodium dodecyl sulfate-PAGE, purified cystathionine gamma-synthase migrated as two molecular species of M(r) 53,000 and 50,000 that are identical in their N-termini. The absorption spectrum obtained at pH 7.5 exhibited a peak at 425 nm due to pyridoxal 5'-phosphate (PLP). The purified enzyme catalyzed the formation of L-cystathionine or L-homocysteine depending on the sulfur-containing substrate, L-cysteine or sulfide. Maximal cystathionine gamma-synthase activity was found at pH 7.4. The apparent Km values for O-phospho-L-homoserine (the unique homoserine ester synthesized in the chloroplast), L-cysteine, and sulfide were 1.4, 0.18, and 0.6 mM, respectively. Inactivation of cystathionine gamma-synthase by DL-propargylglycine (PAG) showed pseudo-first-order kinetics and data were consistent with the existence of an intermediate reversible enzyme-inhibitor complex (Kappi = 140 microM) preceding the formation of a final enzyme-inhibitor complex (kd = 24 x 10(-3) s-1). The irreversibility of the inhibition and the partial restoration of the activity by pyridoxal-phosphate suggest that PAG interacts with the PLP prosthetic group of the enzyme. Kinetic and equilibrium binding studies showed that PAG binding to PLP was considerably enhanced in the enzyme binding pocket compared to that with PLP free in solution.
胱硫醚γ-合酶是甲硫氨酸生物合成途径中的首个特异性酶，已从菠菜叶叶绿体中纯化至表观均一。开发了一种基于L-胱硫醚邻苯二甲醛衍生化和荧光检测的非放射性测定法，以测定胱硫醚γ-合酶活性。在叶绿体的基质部分发现了一种独特的胱硫醚γ-合酶活性，而转硫途径中的第二种酶胱硫醚β-裂解酶则与叶绿体和胞质部分相关（见配套论文）。纯化后的酶比活性为13 U mg-1。通过凝胶过滤和非变性条件下的聚丙烯酰胺凝胶电泳（PAGE），随后进行活性染色估计，天然酶的表观分子量为215,000。基于十二烷基硫酸钠-PAGE，纯化的胱硫醚γ-合酶以分子量为53,000和50,000的两种分子形式迁移，它们的N端相同。在pH 7.5下获得的吸收光谱由于磷酸吡哆醛（PLP）在425 nm处有一个峰值。纯化后的酶根据含硫底物L-半胱氨酸或硫化物催化L-胱硫醚或L-高半胱氨酸的形成。在pH 7.4时发现胱硫醚γ-合酶活性最高。O-磷酸-L-高丝氨酸（叶绿体中合成的唯一高丝氨酸酯）、L-半胱氨酸和硫化物的表观Km值分别为1.4、0.18和0.6 mM。DL-炔丙基甘氨酸（PAG）对胱硫醚γ-合酶的失活表现出假一级动力学，数据与在最终酶-抑制剂复合物形成之前存在中间可逆酶-抑制剂复合物（Kappi = 140 μM）一致（kd = 24×10-3 s-1）。抑制作用的不可逆性以及磷酸吡哆醛对活性的部分恢复表明PAG与酶的PLP辅基相互作用。动力学和平衡结合研究表明，与溶液中游离的PLP相比，PAG在酶结合口袋中与PLP的结合显著增强。
