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Metabolism and Enzymology

Status of the Substrate Binding Sites of Ribulose Bisphosphate Carboxylase as Determined with 2-C-Carboxyarabinitol 1,5-Bisphosphate ¹

Department of Biochemistry, University of Arizona, Tucson, Arizona 85721, Department of Plant Science, University of Arizona, Tucson, Arizona 85721

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 CO₂ and Mg²⁺, were studied. With fully active purified spinach (Spinacia oleracea) Rubisco the rate of tight binding of [¹⁴C]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 Mg²⁺. Addition of [¹⁴C]CABP and EDTA stopped binding of Mg²⁺ and allowed tight binding of the radiolabel only to sites which were CO₂/Mg²⁺-activated at that moment. This approach estimated the amount of CO₂/Mg²⁺-activated sites in the presence of inactive sites and carbamylated sites lacking Mg²⁺. The rate of CO₂ fixation was proportional to the CO₂/Mg²⁺-activated sites. During light-dependent CO₂ 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 Mg²⁺ and CO₂. Lysis of chloroplasts in media with [¹⁴C]CABP plus EDTA estimated those carbamylated sites having Mg²⁺. The loss of Rubisco activation during illumination was partially due to the lack of Mg²⁺ to stabilize the carbamylated sites.

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