Kumaran Sangaralingam, Yi Hankuil, Krishnan Hari B, Jez Joseph M
Donald Danforth Plant Science Center, St. Louis, Missouri 63132, USA.
J Biol Chem. 2009 Apr 10;284(15):10268-75. doi: 10.1074/jbc.M900154200. Epub 2009 Feb 11.
Macromolecular assemblies play critical roles in regulating cellular functions. The cysteine synthase complex (CSC), which is formed by association of serine O-acetyltransferase (SAT) and O-acetylserine sulfhydrylase (OASS), acts as a sensor and modulator of thiol metabolism by responding to changes in nutrient conditions. Here we examine the oligomerization and energetics of formation of the soybean CSC. Biophysical examination of the CSC by size exclusion chromatography and sedimentation ultracentrifugation indicates that this assembly (complex M(r) approximately 330,000) consists of a single SAT trimer (trimer M(r) approximately 110,000) and three OASS dimers (dimer M(r) approximately 70,000). Analysis of the SAT-OASS interaction by isothermal titration calorimetry reveals negative cooperativity with three distinct binding events during CSC formation with K(d) values of 0.3, 7.5, and 78 nm. The three binding events are also observed using surface plasmon resonance with comparable affinities. The stability of the CSC derives from rapid association and extremely slow dissociation of OASS with SAT and requires the C terminus of SAT for the interaction. Steady-state kinetic analysis shows that CSC formation enhances SAT activity and releases SAT from substrate inhibition and feedback inhibition by cysteine, the final product of the biosynthesis pathway. Cysteine inhibits SAT and the CSC with K(i) values of 2 and 70 microm, respectively. These results suggest a new model for the architecture of this regulatory complex and additional control mechanisms for biochemically controlling plant cysteine biosynthesis. Based on previous work and our results, we suggest that OASS acts as an enzyme chaperone of SAT in the CSC.
大分子组装体在调节细胞功能中发挥着关键作用。由丝氨酸O - 乙酰转移酶（SAT）和O - 乙酰丝氨酸巯基化酶（OASS）结合形成的半胱氨酸合酶复合体（CSC），通过响应营养条件的变化，充当硫醇代谢的传感器和调节剂。在此，我们研究了大豆CSC的寡聚化和形成能量学。通过尺寸排阻色谱法和沉降超速离心法对CSC进行生物物理检测表明，该组装体（复合体相对分子质量约为330,000）由一个SAT三聚体（三聚体相对分子质量约为110,000）和三个OASS二聚体（二聚体相对分子质量约为70,000）组成。通过等温滴定量热法分析SAT - OASS相互作用，发现在CSC形成过程中有三个不同的结合事件呈现负协同性，解离常数（K(d)）值分别为0.3、7.5和78纳米。使用表面等离子体共振也观察到了这三个具有可比亲和力的结合事件。CSC的稳定性源于OASS与SAT的快速缔合和极其缓慢的解离，并且这种相互作用需要SAT的C末端。稳态动力学分析表明，CSC的形成增强了SAT的活性，并使SAT从底物抑制和生物合成途径终产物半胱氨酸的反馈抑制中释放出来。半胱氨酸分别以2和70微摩尔的抑制常数（K(i)）抑制SAT和CSC。这些结果为这种调节复合体的结构提出了一个新模型，并为生物化学控制植物半胱氨酸生物合成提供了额外的控制机制。基于先前的工作和我们的结果，我们认为在CSC中OASS充当SAT的酶伴侣。
