title: Status of the substrate binding sites of ribulose bisphosphate carboxylase as determined with 2-C-carboxyarabinitol 1,5-bisphosphate.
abstract: The properties of the tight and specific binding of 2-C-carboxy-d-arabinitol 1,5-bisphosphate (CABP), which occurs only to reaction sites of ribulose 1,5-bisphosphate carboxylase (Rubisco) that are activated by CO(2) and Mg(2+), were studied. With fully active purified spinach (Spinacia oleracea) Rubisco the rate of tight binding of [(14)C]CABP fit a multiple exponential rate equation with half of the sites binding with a rate constant of 40 per minute and the second half of the sites binding at 3.2 per minute. This suggests that after CABP binds to one site of a dimer of Rubisco large subunits, binding to the second site is considerably slower, indicating negative cooperativity as previously reported (S Johal, BE Partridge, R Chollet [1985] J Biol Chem 260: 9894-9904). The rate of CABP binding to partially activated Rubisco was complete within 2 to 5 minutes, with slower binding to inactive sites as they formed the carbamate and bound Mg(2+). Addition of [(14)C]CABP and EDTA stopped binding of Mg(2+) and allowed tight binding of the radiolabel only to sites which were CO(2)/Mg(2+)-activated at that moment. This approach estimated the amount of CO(2)/Mg(2+)-activated sites in the presence of inactive sites and carbamylated sites lacking Mg(2+). The rate of CO(2) fixation was proportional to the CO(2)/Mg(2+)-activated sites. During light-dependent CO(2) fixation with isolated spinach chloroplasts, the amount of carbamylation was proportional to Rubisco activity either initially upon lysis of the plastids or following total activation with Mg(2+) and CO(2). Lysis of chloroplasts in media with [(14)C]CABP plus EDTA estimated those carbamylated sites having Mg(2+). The loss of Rubisco activation during illumination was partially due to the lack of Mg(2+) to stabilize the carbamylated sites.
authors: Department of Biochemistry, University of Arizona, Tucson, Arizona 85721.
authors: G
authors: G
authors: Zhu
authors: R G
authors: RG
authors: Jensen