Exchange Properties of the Activator CO(2) of Spinach Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

The exchange properties of the activator CO₂ of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase were characterized both in vitro with the purified enzyme, and in situ within isolated chloroplasts. Carboxyarabinitol-1,5-bisphosphate, a proposed reaction intermediate analog for the carboxylase activity of the enzyme, was used to trap the activator CO₂ on the enzyme both in vitro and in situ. Modulation of ribulose-1,5-bisphosphate carboxylase/oxygenase activity in intact chloroplasts during a light/dark cycle was associated with a similar modulation in carboxyarabinitol-1,5-bisphosphate-trapped CO₂. The exchange kinetics of the activator CO₂ were monitored by activation of the enzyme to steady state in the presence of ¹²CO₂, followed by addition of ¹⁴CO₂ and determination of the amount of labeled CO₂ trapped on the enzyme by carboxyarabinitol-1,5-bisphosphate. Rate constants (K_obs) for exchange with both the purified enzyme (0.45 min⁻¹) and in illuminated chloroplasts (0.18 min⁻¹) were comparable to the observed rate constants for enzyme activation under the two conditions. A similar exchange of the activator CO₂ was not observed in chloroplasts in the dark. Kinetic analysis of the exchange properties of the purified enzyme were consistent with an equilibrium between active and inactive forms of the enzyme during steady state activation.