Catalysis by cyanobacterial ribulose-bisphosphate carboxylase large subunits in the complete absence of small subunits.
An expression plasmid incorporating the structural gene for the large subunit of a cyanobacterial ribulose-bisphosphate carboxylase, but not the gene for its complementary small subunit, directs the synthesis of large subunits in Escherichia coli. This provides a means for obtaining a preparation of large subunits completely devoid of small subunits, which is not otherwise achievable. In extracts, these large subunits were found predominantly in the form of octamers, but intersubunit interactions were weaker than in the holoenzyme, which contains eight small subunits as well as eight large subunits, and tended to be broken by procedures which separated octamers from lower oligomers and monomers. However, partial purification by anion-exchange chromatography was possible. The large subunits recognized the reaction-intermediate analog, 2'-carboxy-D-arabinitol 1,5-bisphosphate, thus enabling measurement of catalytic site concentrations, but the binding was much weaker than to the holoenzyme. E. coli-produced large subunits catalyzed carboxylation with a kcat of 1% of that of the holoenzyme and the substrate affinities were 3- to 5-fold weaker. They also assembled with heterologous small subunits isolated from spinach ribulose-P2 carboxylase with a 100-fold increase in catalytic activity under standard assay conditions. Since catalysis can proceed in their absence, the small subunits cannot be directly involved in the catalytic chemistry. Their stimulative influence upon catalysis must be exerted by conformational means.
D002384:Catalysis; D002499:Centrifugation, Density Gradient; D011232:Chemical Precipitation; D002845:Chromatography; D000458:Cyanobacteria / Q000201:enzymology*; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D046911:Macromolecular Substances; D010100:Oxygen / Q000494:pharmacology; D010428:Pentosephosphates / Q000378:metabolism; D010957:Plasmids; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013402:Sugar Alcohols
D004274:DNA, Recombinant; D046911:Macromolecular Substances; D010428:Pentosephosphates; D013402:Sugar Alcohols; C032687:2-carboxyarabinitol 1,5-biphosphate; D012273:Ribulose-Bisphosphate Carboxylase; D010100:Oxygen