Fitchen J H, Knight S, Andersson I, Branden C I, McIntosh L
Michigan State University-Department of Energy Plant Research Laboratory, East Lansing 48824.
Proc Natl Acad Sci U S A. 1990 Aug;87(15):5768-72. doi: 10.1073/pnas.87.15.5768.
To explore the role of individual residues in the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), small subunits with single amino acid substitutions in three regions of relative sequence conservation were produced by directed mutagenesis of the rbcS gene from Anabaena 7120. These altered small subunits were cosynthesized with large subunits (from an expressed Anabaena rbcL gene) in Escherichia coli. Mutants were analyzed for effects on quaternary structure and catalytic activity. Changing Glu-13S (numbering used is that of the spinach enzyme) to Val, Trp-67S to Arg, Pro-73S to His, or Tyr-98S to Asn prevented accumulation of stable holoenzyme. Interpretation of these results using a model for the three-dimensional structure of the spinach enzyme based on x-ray crystallographic data suggests that our small subunit mutants containing substitutions at positions 13S and 67S probably do not assemble because of mispairing or nonpairing of charged residues on the interfacing surfaces of the large and small subunits. The failure of small subunits substituted at positions 73S or 98S to assemble correctly may result from disruption of intersubunit or intrasubunit hydrophobic pockets, respectively.
为了探究核酮糖-1,5-二磷酸羧化酶/加氧酶（EC 4.1.1.39）小亚基中单个残基的作用，通过对鱼腥藻7120的rbcS基因进行定向诱变，产生了在三个相对序列保守区域具有单个氨基酸替换的小亚基。这些改变的小亚基与大亚基（来自表达的鱼腥藻rbcL基因）在大肠杆菌中共合成。分析了突变体对四级结构和催化活性的影响。将Glu-13S（使用的编号是菠菜酶的编号）替换为Val、Trp-67S替换为Arg、Pro-73S替换为His或Tyr-98S替换为Asn会阻止稳定全酶的积累。基于X射线晶体学数据，利用菠菜酶三维结构模型对这些结果进行解释表明，我们在13S和67S位置含有替换的小亚基突变体可能由于大亚基和小亚基界面表面带电残基的错配或不配对而无法组装。在73S或98S位置替换的小亚基未能正确组装，可能分别是由于亚基间或亚基内疏水口袋的破坏。
