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结构域，通过这些结构域来决定组装和活性。核酮糖-1，5-二磷酸羧基/加氧酶(Rubisco)及其两个亚基LSU和SSU是研究这种亚基组装和相互作用的一个很好的模型。广泛的生物化学和生理学研究，包括蛋白质/基因序列和晶体结构。在L8S8 Rubisco全酶中，Rubisco SSU不贡献任何活性残基，是活性所必需的。本项目将集中在SSU结构域在决定i)SSU折叠，ii)活性L8S8的组装，以及III)SSU动力学参数的影响方面的功能。其中两个方面已经被我们广泛研究(Wasmann等人，1989；Ramage，1990；Smrcka等人，1990 a，b)，为我们将通过定点突变引入SSU的特定突变提供了基础。经过定点突变后，SSU基因将在细菌中表达，并在体外与可溶性蓝藻L8组装。修饰的SSU的组装也将在转运到叶绿体后进行研究。由同源和异源亚基组成的全酶将测量底物的激活动力学、底物和效应物结合的稳定性以及底物类似物的结合参数。由于LSU在没有SSU的情况下极难溶解，因此阻止了对高等植物Rubrisco的类似研究。因此，改变后的SSU将在体外、在分离的叶绿体中表达和组装，并在体内通过核酶基因构建失活驻留的SSU表达。(作为建立核酶功能的中间步骤，核酶结构和突变的SSU将在电穿孔基因进入叶肉原生质体后在体外进行测试)。将测定改良的SSU对高等植物Rubisco活性的几个参数的影响。假设蓝藻和高等植物中的SSU补偿底物与活性部位结合所引起的构象变化。

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.