Functional Domains in Rubisco SSU

多聚体酶的亚基已经进化出相互作用的蛋白质 决定组装和活性的结构域。 核酮糖- 1，5-二磷酸羧化酶/加氧酶（rubisco），其两个 亚单位，LSU和SSU，是一个很好的模型，研究这种 亚基组装和相互作用。 广泛的生物化学和 生理学研究可用，包括蛋白质/基因 序列和晶体结构。 Rubisco SSU， 不贡献任何活性位点残基，是必需的， L 8 S8 Rubisco全酶活性。 该项目将重点 关于SSU中结构域在确定i）折叠 SSU，ii）活性L 8 S8的组装，和iii）活性L 8 S8的SSU影响， 动力学参数 其中两个方面已被广泛 由我们研究（Wasmann等人，1989; Ramage，1990; Smrcka等人， 1990 a，B）提供了特定突变的基础，我们将 通过定点诱变引入SSU。 现场后- 定向诱变后，SSU基因将在细菌中表达， 与可溶性蓝藻L 8的体外组装。 大会 修改后的SSU也将在运输到 叶绿体 活化动力学、底物稳定性和 效应物结合和底物类似物的结合参数 将用由同源和 异源亚基 高等植物Rubisco的类似研究 由于LSU在不存在 特别调查组 因此，改变的SSU将在细胞中表达和组装。 体外，在分离的叶绿体中，和体内， 核酶基因对SSU基因表达失活作用 构建体 (As一个中间步骤，以建立 核酶、核酶构建体和突变的SSU的功能性 将在体外进行测试，在将基因电穿孔到 叶肉原生质体）。 改性SSU对几种 高等植物Rubisco的活性参数将是 测定 假设是SSU（蓝细菌和 高等植物）补偿由蛋白质诱导的构象变化。 底物与活性位点的结合。

Subunits of multimeric enzymes have evolved interactive protein domains by which assembly and activity are determined. Ribulose- 1,5-bisphosphate carboxylase/oxygenase (rubisco), with its two subunits, LSU and SSU, is an excellent model for studying such subunit assembly and interactions. Extensive biochemical and physiological studies are available, including protein/gene sequences and crystallographic structures. Rubisco SSU, which does not contribute any active site residues, is essential in the L8S8 rubisco holoenzyme for activity. This project will focus on the functions of domains in SSU in determining i) folding of SSU, ii) assembly of active L8S8, and iii) SSU influence of kinetic parameters. Two of these aspects have been extensively studied by us (Wasmann et al., 1989; Ramage, 1990; Smrcka et all, 1990a,b) providing the basis for specific mutations which we will introduce into SSU by site directed mutagenesis. After site- directed mutagenesis, SSU genes will be expressed in bacteria and assembled with soluble cyanobacterial L8 in vitro. Assembly of modified SSU will also be investigated following transport into chloroplast. Kinetics of activation, stability of substrate and effector binding, and binding parameters of substrate analogs will be measured with holoenzymes composed of homologous and heterologous subunits. Similar studies on higher plant rubisco are prevented as LSU is extremely insoluble in the absence of SSU. Therefore, altered SSU will be expressed and assembled in vitro, in isolated chloroplasts, and in vivo, following inactivation of resident SSU expression by a ribozyme gene construct. (As an intermediary step to establishing the functionality of ribozymes, ribozyme constructs and mutated SSU will be tested in vitro, after electroporation of genes into mesophyll protoplasts). The effect of modified SSU on several parameters of activity of higher plant rubisco will be determined. The hypothesis is that SSU (cyanobacteria and in higher plants) compensates conformational changes induced by the binding of substrate to the active site.