Modulation of the tight binding of carboxyarabinitol 1,5-bisphosphate to the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase.
The large subunit (L) of ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) from Synechococcus PCC 6301 was expressed in Escherichia coli, purified as the octamer L8, and analyzed for its ability to tightly bind the transition state analog, 2-carboxyarabinitol 1,5-bisphosphate (CABP). [14C]CABP remained tightly bound to L8 after challenging with [12C]CABP and gel filtration, indicating that L8 alone without the small subunit (S) could tightly bind CABP. Binding of CABP to L8 induced a shift in the gel filtration profile due to apparent aggregation of L8. Aggregation did not occur with the L8S8-CABP complex nor with L8-CABP in the presence of 150 mM MgCl2. If ionic strength was increased with either KCl or MgCl2 during or after the binding of [14C]CABP to L8, [14C]CABP in the complex exchanged with [12C]CABP and was lost from the protein. Ionic strength strongly affected the rate constant (k4) for [14C]CABP dissociation from the L8-[14C]CABP complex, but had little effect on k4 for the L8S8-CABP complex. The differences in CABP binding characteristics between the L8-CABP and L8S8-CABP complexes demonstrate that S is intimately involved in maintaining the stability of the tight binding of CABP to the active site. These are the same interactions stabilizing the intermediate, 3-keto-2-carboxyarabinitol 1,5-bisphosphate, to native rubisco during CO2 fixation.
Smrcka
A V
AV
Department of Biochemistry, University of Arizona, Tucson 85721.
Bohnert
H J
HJ
Jensen
R G
RG
D001665:Binding Sites; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000201:enzymology*; D004926:Escherichia coli / Q000235:genetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010428:Pentosephosphates / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013402:Sugar Alcohols / Q000378:metabolism*
D046911:Macromolecular Substances; D010428:Pentosephosphates; D011994:Recombinant Proteins; D013402:Sugar Alcohols; C032687:2-carboxyarabinitol 1,5-biphosphate; D012273:Ribulose-Bisphosphate Carboxylase